Systems and methods for serial flow emulsion processes

ABSTRACT

Disclosed herein are systems and methods for serial flow emulsion processes. Systems and methods as described herein result in reduced cross-contamination.

CROSS-REFERENCE

This application is a divisional of U.S. patent application Ser. No.17/062,040 filed Oct. 2, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/372,290, filed Apr. 1, 2019, which claims thebenefit of U.S. Provisional Application No. 62/651,619, filed Apr. 2,2018;

This application is also a divisional of U.S. patent application Ser.No. 17/021,876 filed Sep. 15, 2020, which is a continuation of U.S.patent application Ser. No. 16/372,290, filed Apr. 1, 2019, which claimsthe benefit of U.S. Provisional Application No. 62/651,619, filed Apr.2, 2018; and

This application is also a divisional of U.S. application Ser. No.17/021,884 filed Sep. 15, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/372,290, filed Apr. 1, 2019, which claims thebenefit of U.S. Provisional Application No. 62/651,619, filed Apr. 2,2018.

BACKGROUND

Serial flow emulsion systems processes have numerous applications inphysical, chemical, and biological areas, and improvements in suchsystems and processes are useful. For example, the quantitation ofnucleic acids is an indispensable technique in medical and biologicalapplications. Methods for detecting and quantitating nucleic acids, suchas emulsion-based digital nucleic acid amplification, includingemulsion-based polymerase chain reaction (PCR), provide greater accuracyand convenience as compared to traditional nucleic acid amplification,such as traditional polymerase chain reaction (PCR) methods. Performingemulsion-based digital nucleic acid amplification in serial-flow,however, face problems with cross-contamination between individualvolumes of the dispersed phase and/or the channel and/or tube containingthe emulsion.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an intake system with a waste.

FIG. 2 shows an intake system with blowback.

FIG. 3 shows an intake system with spacer fluid addition.

FIGS. 4A, 4B, 4C and 4D show fluid “parfait.”

FIGS. 5A and 5B shows a system for cleaning an intake conduit.

FIGS. 6A and 6B shows a system for sampling and cleaning an intakeconduit.

FIG. 7 shows a system for injecting a sample comprising a waste station.

FIGS. 8A and 8B show patterns for sampling to avoid cross-contamination.

FIGS. 9A and 9B show examples of aspirating a fluid.

FIGS. 10A and 10B show examples of injecting a sample from a samplecontainer with a cover.

FIGS. 11A and 11B show systems and methods for creating layered fluidsin sample containers.

FIG. 12 shows a system for sensing the level of a fluid with a samplinginlet.

FIGS. 13A and 13B shows a designs of seals to avoid samplecontamination.

FIGS. 14A and 14B show sealing systems.

FIGS. 15A, 15B, and 15C show systems for aspirating samples and piercingseals.

FIG. 16 shows design of aspiration tip for filtration.

FIGS. 17A and 17B shows methods for aspirating a fluid.

FIG. 18 shows systems for supplying sample fluids and cleaning.

FIG. 19 shows systems for supplying sample fluids and cleaning.

FIG. 20 shows a self-filling system for applying cleaning fluids.

FIG. 21 shows a fluid aspirator.

FIG. 22 shows a system for aspirating fluid.

FIG. 23 shows a fluid aspirator.

FIG. 24 shows a system for aspirating a fluid.

FIG. 25 shows a system with second vertical actuator.

FIG. 26 shows a system for holding a fluid container.

FIGS. 27A and 27C show one system for cleaning a fluid aspirator.

FIGS. 27B and 27D show a second system for cleaning a fluid aspirator.

FIG. 28 shows a system for providing sample and cleaning fluids.

FIG. 29 shows a system for providing fluid reagents.

FIG. 30 shows a cartridge for supplying reagents or collecting waste.

FIG. 31 shows a syringe pump.

FIG. 32 shows a bank of syringe pumps.

FIG. 33 shows an injector system.

FIG. 34 shows an injector with a plurality of common conduits.

FIG. 35 shows a multi-position injector.

FIGS. 36A and 36B show a rotary single face injector in two differentpositions.

FIG. 37 shows a rotary dual face injector.

FIG. 38 shows a reverse-y partitioner, with a 90 degree intersect.

FIGS. 39A and 39B show different angles of intersect in reverse-ypartitioner.

FIG. 40 shows a gravitational arrangement of channels.

FIGS. 41A, 41B, and 41C show different configurations of T-junctionpartitioners.

FIG. 42 shows a cross junction partitioner.

FIGS. 43A, 43B, 43C, and 43D show conduit, e.g., partitionerconnections.

FIGS. 44A and 44B show different partitioner connections to tubing.

FIG. 45 shows partitioner connections for formed channels.

FIGS. 46A and 46B show different methods of manufacturing ofpartitioners.

FIGS. 47A and 47B show removal of additional continuous phase.

FIG. 48 shows recycle of oil with disengagers.

FIG. 49 shows disengagement for droplet slowing.

FIG. 50 shows a star-shaped detector.

FIG. 51 shows a concentric tube separator.

FIG. 52 shows a T-junction chip-based separator.

FIGS. 53A and 53B show two views of an on-chip interrogation region.

FIG. 54 shows a collision-style separator.

FIG. 55 shows a Y-style separator with off-chip detection.

FIGS. 56A and 56B show two views of a constricted tube separator.

FIGS. 57A and 57B show a conduit formed in a substrate. 57A: in a chip;57B: in a conduit.

FIG. 58 shows an interrogation region.

FIG. 59 shows a tubular interrogation region with opposingexcitation/detection.

FIG. 60 shows a tubular interrogation region with in-pathexcitation/detection.

FIG. 61 shows on-chip interrogation region with opposingexcitation/detection.

FIGS. 62A and 62B show use of an optical restriction to limit acceptedelectromagnetic radiation to a single partition.

FIG. 63 shows positive signal detection.

FIG. 64 shows negative signal detection.

FIGS. 65A and 65B show a multiexcitation source. 65A: not foldedaperture; 65B: folded aperture.

FIG. 66 shows temporal modulation.

FIGS. 67A, 67B and 67C show different configurations of tubularspectrometer arrangements with diffraction.

FIGS. 68A and 68B show a tubular spectrometer arrangements withdiffraction.

FIG. 69 shows a tubular spectrometer with turning mirror.

FIG. 70 shows offset excitation sources.

FIG. 71 shows lock-in detection for partitions.

FIGS. 72A and 72B show blank sample discrimination.

FIG. 73 shows fiber excitation on tube.

FIG. 74 shows two detector sample discrimination.

FIG. 75 shows a star-shaped detector.

FIG. 76 shows a cylindrical heater-reactor.

FIGS. 77A and 77B show a heater-reactor conduit.

FIG. 78 shows a three temperature zone heater-reactor.

FIG. 79 shows a four temperature zone heater-reactor.

FIG. 80 shows a discrete four temperature zone heater-reactor.

FIGS. 81A, 81B and 81C show a gradient heater-reactor.

FIGS. 82A and 82B show a 2-step PCR heater-reactor.

FIGS. 83A and 83B show a RT-PCR heater-reactor.

FIG. 84 shows a system diagram comprising a first dispersed phase, asecond dispersed phase, a sampling device (“sampler”), a continuousphase reservoir, an injector, a reactor, and a detector.

FIG. 85 shows a system diagram comprising a first dispersed phase, asecond dispersed phase, a third dispersed phase, a sampling device(“sampler”), a continuous phase reservoir, an injector, a reactor, and adetector.

FIG. 86 shows a system diagram comprising a first dispersed phase, asecond dispersed phase, a third dispersed phase, a fourth dispersedphase a sampling device (“sampler”), a continuous phase reservoir, aninjector, a reactor, and a detector.

FIG. 87 shows a diagram comprising a sampler, pump, continuous phase,and inject

FIG. 88 shows a diagram comprising a sampler, pump, continuous phase,and inject

FIG. 89 shows a sampler intake comprising a sharp, hard tube forbreaking through a seal and a sampling tube.

FIG. 90 shows a diagram of a detector.

FIG. 91 shows a detailed arrangement of FIG. 90.

FIG. 92 shows a diagram of a detector comprising a pinhole.

FIG. 93 shows a diagram of a system for achieving multiplexing.

FIG. 94 show a diagram of a system for achieving multiplexing.

DETAILED DESCRIPTION I. Overview II. Intake System III. Injector IV.Process System

A. Partitioner

B. Reactor

C. Detector

V. Definitions VI. Numbered Embodiments I. Overview

Systems and methods provided herein relate to flowing emulsions.

In certain embodiments, provided herein are systems and methodscomprising an intake system and a process system. An emulsion is formedand flows through the process system to be processed. The emulsioncomprises partitions of a dispersed phase in a continuous phase;typically, the dispersed phase is supplied by the intake system, e.g.,as a sample or portion of a sample that is taken up by the intakesystem, and continuous phase is supplied, at least in part, by theprocess system. In certain embodiments, the intake system and theprocess system are separate, e.g., at no time is there continuous flowbetween the intake system and the process system.

Systems and methods can include use of an injector, where the injectoris positioned between the intake system and the process system, and theinjector can be in fluid communication with the intake system, or influid communication with the process system, but not bothsimultaneously. A series of aliquots of dispersed phase, e.g., a seriesof aliquots from samples or portions of samples, can be flowed throughthe intake system into the injector, then each is injected separatelyinto the process system. Methods can include flowing one or more of apurge fluid, a denaturing fluid, and/or a spacer fluid through theintake system, e.g., including the injector, such as methods asdescribed herein, between flow of aliquots of dispersed phase, e.g.,sample, through the intake system, e.g., including the injector, such asbetween injections of dispersed phase into the process system. Theinjector can be configured to inject a fixed volume from the intakesystem into the process system, for example, a volume of 0.1-200 uL,such as 0.1-100 uL, for example, 1-100 uL.

The process system can include a partitioner (also referred to as adroplet generator herein) for partitioning dispersed phase supplied bythe intake system, e.g., a sample or portion of a sample comprisingdispersed phase, into partitions in a continuous phase, e.g., forming anemulsion. Any suitable partitioner, such as partitioners describedherein, may be used. The partitioner can have at least one inlet fordispersed phase, at least one inlet for continuous phase, and an outletleading to the rest of the process system. In certain embodiments, thepartitioner comprises a “reverse-y” partitioner, as described furtherherein. In certain embodiments, the partitioner is relativelyinsensitive to flow variations in the inlets, as described furtherherein. In certain embodiments, the partitioner comprises an inlet fordispersed phase and an inlet for continuous phase that comprise conduitsthat meet at an angle of 170-180 degrees, for example, at an angle of180 degrees (co-axial), as described further herein. In certainembodiments, the partitioner can produce partitions of an average volumebetween 0.05 and 50 nL, such as between 0.1 and 10 nL.

The process system can further comprise a reactor for initiating ormodulating a reaction in the partitions. The reactor can be any suitablereactor, such as reactors as described herein. In certain embodiments,the reactor comprises a thermal cycler, e.g., for performing polymerasechain reaction (PCR). In certain embodiments the reactor comprises aheating core maintained at a consistent temperature, e.g., forperforming incubations. In certain embodiments, systems and methodsinclude an intake system, a process system, an injector positionedbetween the intake system and the process system where the injector canbe in fluid communication with the intake system, in fluid communicationwith the process system, but not both simultaneously, and a reactor,such as a reactor comprising a thermal cycler. Processes are generallydescribed in terms of PCR herein, however, any suitable process may beconducted in the process system, including but not limited to sampleprocessing applications including cell lysis, cell growth, ligation,digestions, nucleic acid assembly reactions, nucleic acid editing,nucleic acid modification, or sample analysis including the detection ofnucleic acids, proteins, and microbial organisms using reactions includebut are not limited to RNA transcription, hybridization chain reaction(HCR), nicking chain reaction, loop-mediated isothermal amplification(LAMP), strand displacement amplification (SDA), helicase-dependentamplification (HDA), nicking enzyme amplification reaction (NEAR),protein detection, protein melt temperature analysis, small moleculedetection, microbial growth rate testing, antibiotic resistance testing,microbial small molecule production, molecule-molecule interactionstudies. In certain embodiments systems and methods provide an intakesystem, an injector, where the injector is positioned between the intakesystem and the process system, and the injector can be in fluidcommunication with the intake system, or in fluid communication with theprocess system, but not both simultaneously, a partitioner, such as apartitioner described above or elsewhere herein, and a reactor. Incertain embodiments, at least a portion of partitions formed by thepartitioner comprise at least one nucleic acid and the reactor is athermal cycler for performing PCR on the partitions.

The process system can comprise a detector for detecting one or morecharacteristics of partitions as they flow through the detector. Thedetector can be any suitable detector, such as a detector as describedherein. Partitions flow through the detector in single file in a conduitthat includes an interrogation region where, e.g. electromagneticradiation from the flow through the interrogation region, such aselectromagnetic radiation from a partition flowing through theinterrogation region, is emitted to be detected by one or more detectionelements. The detector can be configured so that electromagneticradiation from partitions that is detected by the detection element all,or substantially all, comes from individual partitions as they flowthrough the interrogation region; that is, there is little or no overlapin detected electromagnetic radiation from one partition to another. 1)In certain embodiments, the detector comprises an optical restrictionconfigured and positioned between the interrogation region and thedetection element so that only a portion of electromagnetic radiationfrom the interrogation region that could otherwise be detected by thedetection element is detected, for example, less than 10% of theelectromagnetic radiation, such as less than 1%. In certain embodiments,systems and methods include an intake system, a process system, aninjector positioned between the intake system and the process systemwhere the injector can be in fluid communication with the intake system,in fluid communication with the process system, but not bothsimultaneously, where the process system comprises a detector comprisingan optical restriction. In certain embodiments, systems and methodsinclude a partitioner and a detector, where the detector comprises anoptical restriction. 2) In certain embodiments, the region of theconduit in the interrogation region has a cross-sectional area that isequal to or less than the average spherical cross-sectional area ofpartitions flowing through the detector, such as less than 90% or lessthan 50%. In certain embodiments, systems and methods include an intakesystem, a process system, an injector positioned between the intakesystem and the process system where the injector can be in fluidcommunication with the intake system, in fluid communication with theprocess system, but not both simultaneously, where the process systemcomprises a detector comprising a conduit comprising an interrogationregion where the region of the conduit in the interrogation region has across-sectional area that is equal to or less than the average sphericalcross-sectional area of partitions flowing through the detector, such asless than 95%, or 90% or less than 50%. In certain embodiments, systemsand methods include a partitioner and a detector, where the detectorcomprising a conduit comprising an interrogation region where the regionof the conduit in the interrogation region has a cross-sectional areathat is equal to or less than the average spherical cross-sectional areaof partitions flowing through the detector, such as less than 90% orless than 50%. 3) In certain embodiments the detector comprises anexcitation source, or a plurality of excitation sources, such as atleast 2, 3, 4, or 5 excitation sources, for supplying electromagneticradiation to the interrogation region, where the excitation source orsources comprise a lock-in amplification system. In such systems only asingle detection element, e.g., photodetection element, such as asilicon photomultiplier, may be used, even with a plurality ofexcitation sources. In certain embodiments, systems and methods includean intake system, a process system, an injector positioned between theintake system and the process system where the injector can be in fluidcommunication with the intake system, in fluid communication with theprocess system, but not both simultaneously, where the process systemcomprises a detector and the detector comprises an excitation source, ora plurality of excitation sources, such as at least 2, 3, 4, or 5excitation sources, for supplying electromagnetic radiation to theinterrogation region, where the excitation source or sources comprise alock-in amplification system; in certain embodiments, only a singledetection element is used. 4) In certain embodiments the detectorcomprises a partition separation system that separates partitions beforethey reach the interrogation region, e.g., by adding continuous phasebetween partitions before they reach the interrogation region. Incertain embodiments, systems and methods include an intake system, aprocess system, an injector positioned between the intake system and theprocess system where the injector can be in fluid communication with theintake system, in fluid communication with the process system, but notboth simultaneously, where the process system comprises a detector thatcomprises a partition separation system that separates partitions beforethey reach the interrogation region, e.g., by adding continuous phasebetween partitions before they reach the interrogation region. 5)Systems and methods provided herein may also include one or moredisengager, e.g., a system that removes continuous phase from anemulsion, for example, after a partitioner but prior to a reactor, orafter a detector, or both, and, in certain embodiments, adds back someor all of the removed continuous phase to the process system, e.g., at apartition separation system. In certain embodiments, systems and methodsinclude an intake system, a process system, an injector positionedbetween the intake system and the process system where the injector canbe in fluid communication with the intake system, in fluid communicationwith the process system, but not both simultaneously, where the processsystem comprises one or more disengager, e.g., a system that removescontinuous phase from an emulsion, for example, after a partitioner butprior to a reactor, or after a detector, or both, and, in certainembodiments, adds back some or all of the removed continuous phase tothe process system, e.g., at a partition separation system. 6) Incertain embodiments the conduit of the interrogation region isconfigured to have the same or substantially the same transmittance,e.g., for electromagnetic radiation from excitation sources that reachesthe interrogation region and for electromagnetic radiation from theinterrogation region that is detected by the detection element, aroundthe circumference of the conduit; for example, the conduit can be atube, such as a tube with a circular or substantially circularcross-section. Such a configuration can allow for, e.g., coplanar orsubstantially coplanar arrangement of a plurality of excitation sources,such as at least 2, 3, 4, or 5 excitation sources, and/or one or moredetection elements, such as in a plane orthogonal or substantiallyorthogonal to an axis of flow of partitions in the interrogation region.In certain embodiments, systems and methods include an intake system, aprocess system, an injector positioned between the intake system and theprocess system where the injector can be in fluid communication with theintake system, in fluid communication with the process system, but notboth simultaneously, where the process system comprises a detectorcomprising an interrogation region comprising a conduit, where theconduit of the interrogation region is configured to have the same orsubstantially the same transmittance, e.g., for electromagneticradiation from excitation sources that reaches the interrogation regionand for electromagnetic radiation from the interrogation region that isdetected by the detection element, around the circumference of theconduit; for example, the conduit can be a tube, such as a tube with acircular or substantially circular cross-section. In certainembodiments, systems and methods provided herein include a detector withcharacteristics of at least one of 1), 2), 3), 4), 5) and 6). In certainembodiments, systems and methods provided herein include a detector withcharacteristics of at least two of 1), 2), 3), 4), 5) and 6). In certainembodiments, systems and methods provided herein include a detector withcharacteristics of at least three of 1), 2), 3), 4), 5) and 6). Incertain embodiments, systems and methods provided herein include adetector with characteristics of at least four of 1), 2), 3), 4), 5) and6). In certain embodiments, systems and methods provided herein includecharacteristics of at least five of 1), 2), 3), 4), 5) and 6). Incertain embodiments, systems and methods provided herein include adetector with characteristics of all of 1), 2), 3), 4), 5) and 6). Incertain embodiments, systems and methods provided herein include anintake system and a process system, wherein the intake system and theprocess system are never in fluid communication, and the process systemcomprises a detector with characteristics of at least one of 1), 2), 3),4), 5) and 6). In certain embodiments, systems and methods providedherein include an intake system and a process system, wherein the intakesystem and the process system are never in fluid communication, and theprocess system comprises a detector with characteristics of at least twoof 1), 2), 3), 4), 5) and 6). In certain embodiments, systems andmethods provided herein include an intake system and a process system,wherein the intake system and the process system are never in fluidcommunication, and the process system comprises a detector withcharacteristics of at least three of 1), 2), 3), 4), 5) and 6). Incertain embodiments, systems and methods provided herein include anintake system and a process system, wherein the intake system and theprocess system are never in fluid communication, and the process systemcomprises a detector with characteristics of at least four of 1), 2),3), 4), 5) and 6). In certain embodiments, systems and methods providedherein include an intake system and a process system, wherein the intakesystem and the process system are never in fluid communication, and theprocess system comprises a detector with characteristics of at leastfive of 1), 2), 3), 4), 5) and 6). In certain embodiments, systems andmethods provided herein include an intake system and a process system,wherein the intake system and the process system are never in fluidcommunication, and the process system comprises a detector withcharacteristics of all of 1), 2), 3), 4), 5) and 6).

Components of the systems are fluidly connected as appropriate and asdescribed further herein. Generally, the intake side can comprises acontinuous conduit between an intake tip and the injector; where theinjector comprises a common conduit that is configured to be fluidlyconnected to the intake side at a first intake system conduit; it willalso be fluidly connected to a second intake system conduit, e.g.,leading to waste. This allows a dispersed phase, e.g., a sample, such asa sample in aqueous phase, to be transported into the common conduit ofthe injector and fill or partially fill the common conduit. The commonconduit can have a fixed volume so that this volume can be injected intothe process side. Surfaces of the intake conduit that come in contactwith dispersed phase comprising a first fluid, transported by the intakesystem, e.g., with sample, such as sample in a aqueous phase, can havegreater affinity for a second fluid that is passed through part or allof the conduit, such as a continuous phase, or such as a purge fluid orother fluid as described herein. In this way, e.g., residual sample canbe displaced from the conduit through, e.g. a cleaning phase. In certainembodiments, the surfaces comprise a fluoropolymer and the second fluidcomprises a fluorinated oil. Surfaces of the injector that come incontact with dispersed phase comprising a first fluid, transported bythe intake system, e.g., with sample, such as sample in a aqueous phase,can have greater affinity for a second fluid that is passed through theinjector, such as a continuous phase, or such as a purge fluid or otherfluid as described herein. In certain embodiments, the surfaces comprisea fluoropolymer and the second fluid comprises a fluorinated oil. Whenthe injector is positioned to be in fluid communication with the processside, there is typically a first process conduit positioned to be influid communication with the common conduit of the injector and a secondprocess conduit in fluid communication with the common conduit, wherethe first process conduit transports a fluid, such as continuous phase,into the injector and displaces the dispersed phase, e.g., sample, suchas sample in an aqueous phase, into the second process conduit where itis transported to the rest of the process system. The second processconduit is generally fluidly connected to further conduits in thesystem, so that dispersed phase transported in the intake side andthrough the injector flows in the conduits from the injector through theprocess system and eventually exits the system. At various points otherconduits may join the main process conduit, for example, at apartitioner where, e.g., continuous phase is added to dispersed phase toproduce an emulsion; in certain embodiments, a partition separationsystem may be used to, e.g., add continuous phase between partitions toseparate the partitions prior to detection. In certain embodiments, flowthrough the conduit from at least a partitioner through the rest of thesystem is continuous; in certain embodiments, flow from the injectorinto the process system is discontinuous, e.g., there are times when theinjector is disconnected from the process system and no flow occursthrough the injector to the process system. Thus, e.g., introduction ofdispersed phase, e.g., sample, into the system may be discontinuous. Forexample, a first aliquot of dispersed phase, e.g., comprising a firstsample, may be introduced into the process system, then a second aliquotof dispersed phase, e.g., comprising a second sample, may be separatelyintroduced into the process system, separated from the first sample.Surfaces of the process system conduit and other conduits and surfacesthat come in contact with dispersed phase comprising a first fluid,transported by the intake system, e.g., with sample, such as sample in aaqueous phase, can have greater affinity for a second fluid that ispassed through the conduit, such as a continuous phase. In certainembodiments, the surfaces comprise a fluoropolymer and the second fluidcomprises a fluorinated oil. In certain embodiments, all or asubstantial portion of surfaces of the system that come in contact witha first fluid, such as a dispersed phase, e.g., a sample such as anaqueous sample, from intake system through injector through processsystem, have greater affinity for a second fluid, e.g., a continuousphase, that is introduced into the system after the first fluid. Incertain embodiments, at least 90, 95, 99, 99.5, 99.9, 99.95, or 99.99%of surfaces of the system have greater affinity for the second fluidthan for the first fluid. In certain embodiments, surfaces comprise afluoropolymer and the second fluid comprises a fluorinated oil.

Dimensions of conduits may be any suitable dimensions, as describedherein. Exemplary dimensions for various components (given as diametersfor a circular cross-section): aspiration tip/intake tip, 0.1-254 um,for example 1-254 um, such as 25-75 um; aspiration line/intake line,10-3175 um, for example, 200-800 um, such as 200-300 um; common conduitof injector, 10-3100 um, for example, 250-750 um, such as 450-550 um;conduit from injector to partitioner, 100-2500 um, for example, 200-500um, such as 200-300 um; inlet to partitioner, 10-2500 um, for example100-500 um, such as 200-300 um; outlet from partitioner, 10-2500 um, forexample 100-500 um, such as 200-300 um; reactor, 10-2500 um, for example100-500 um, such as 200-300 um; interrogation region of detector, 10-250um, for example 75-100 um, such as 80-100 um.

At certain points in the system, a first conduit may be connected to asecond, different conduit. This can occur, e.g., at a junction between aconduit from the injector to a conduit in a partitioner, and/or betweena conduit in a partitioner and a conduit leading to a reactor, and/or bea conduit leading from a reactor to a conduit in a partition separationsystem, and/or between a conduit leading from a partition separationsystem to a conduit in a detector, and/or between a conduit leading froma reactor to a conduit in a detector. Any or all of these connectionsrepresent points where a disruption of flow may occur. In certainembodiments provided herein, connections between a first conduit and asecond, different, conduit, are configured to cause minimal or nodisruption to flow. Such connections are described further herein.

In certain embodiments a dispersed phase is transported by an intakesystem from a container, e.g., a sample container, into the injector,and injected from the injector into the process system, where it movesthrough the process system, e.g., through a partitioner. The dispersedphase may be surrounded by continuous phase at least in the injector,and generally from the start of the intake system, so that when it isinjected in the process system, it comprises dispersed phase incontinuous phase. As the intake system transports a series of dispersedphase aliquots into the injector and ultimately into the process system,the aliquots each form a packet of dispersed phase in continuous phase(also referred to herein as a programmed emulsion) as they areintroduced into the process system, i.e., a first emulsion. The packets(i.e., partitions in this first emulsion) may be any suitable volume,such as volumes described herein, such as 0.1-200 uL, or 0.1-100 uL, or1-50 uL, or 5-50 uL. This volume may be a fixed volume from a fixedvolume in the injector. Each packet can then be further divided into aplurality of partitions in continuous phase at a partitioner, e.g., eachpacket can be further divided into at least 100, 500, 1000, 5000,10,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, or 100,000partitions in continuous phase, i.e., further divided into a secondemulsion, for example, partitions of any suitable volume in continuousphase, such as volumes described herein, for example, an average volumeof 0.05-50 nL, such as 0.1-10 nL, for example, 0.1-1.0 nL. The pluralityof partitions flow in a common conduit through the rest of the system;in certain cases, e.g., when a separation fluid is added prior to adetector, one or more branch conduits may intersect with the mainconduit, e.g., for adding a separation fluid or removing a fluid.

In systems that include an intake side and a process side, where theintake side is used to take up a series of aliquots, e.g., sample and/orother material, from one or more containers, e.g., sample containers,and send them to the process side, problems with contamination, e.g.,cross-contamination, can occur. As used herein, “cross-contamination”includes 1) sample-to-sample carryover; 2) within-sample alterationsthat affect processes occurring on the process side of the system (forexample, coalescence of partitions within a sample); and 3) introductionof material into a sample from the environment, e.g., dust, materialsfrom a user, and the like. In systems and methods provided herein, thesame intake and process system can be used to process a series ofsamples, without the need either to replace portions of either intake orprocess system, or to use separate portions of intake or process systemfor separate samples, and still maintain very low levels ofcross-contamination of samples, for example, an average of less than 20,10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.01, 0.05, or 0.01, or 0.005, or0.001%. Thus, systems and methods provided herein can utilize the samecomponents from sample to sample, e.g., the same intake line, the sameinjector, the same partitioner, the same reactor, and/or the samedetection unit, with little or no cross-contamination of samples, forexample, an average of less than 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.01,0.05, 0.01, 0.005, or 0.001%. One suitable method for measuringcross-contamination is to introduce a first sample in the system thatcontains a high level of molecules of interest, e.g., a high level of anucleic acid, such as DNA, perform normal cleaning routines, thenintroduce a second sample into the system that contains no molecules ofinterest, e.g., no nucleic acid such as DNA. Both samples are processedby the process system, e.g., undergo PCR in the case of a digital PCRsystem. If cross-contamination is zero, then partitions of the secondsample will contain no molecules of interest from the first sample,e.g., no DNA molecules, and should be processed and detected so that nopositive signals are received at the detector. Any partitions of thesecond sample that give a positive signal at the detector, such as asignal indicating nucleic acid amplification, indicatescross-contamination from the first sample to the second sample. Forexample, in a digital PCR system, if a first sample contains 50,000molecules of DNA, and the second sample contains no DNA, all partitionformed from the second sample should give a negative signal (no DNAamplification). If a single partition of the second sample gives apositive signal, it can be assumed that a molecule of DNA from the firstsample has cross-contaminated the second sample, and the level ofcross-contamination is 1/50,000, or 0.002%. If ten partitions of thesecond sample give a positive signal, it can be assumed that 10molecules of DNA from the first sample has cross-contaminated the secondsample, and the level of cross-contamination is 10/50,000, or 0.02%. Forpurposes of this type of assay, it is assumed that a positive signal ina partition of the second sample represents a single molecule ofinterest, e.g., a single DNA molecule, in the partition of the secondsample.

a) Sample-to-sample carryover is one source of cross-contamination. Thatis, remnants of a first sample can carry over into a second, subsequentsample, and/or into further subsequent samples. In certain embodiments,such as in PCR systems, e.g., digital PCR systems, even a singlemolecule from a first sample, e.g., a single nucleic acid, if present ina second sample, can be, e.g., amplified and detected and give a falsepositive in the second sample. Other contaminants can also carry overfrom a first sample, e.g., materials that interfere with one or moreprocesses that occur on the process side. In systems and methodsprovided herein, the same intake and process system can be used toprocess a series of samples, without the need either to replace portionsof either intake or process system, or to use separate portions ofintake or process system for separate samples, and still maintain verylow levels of sample-to-sample carryover between samples. Thus, systemsand methods provided herein can utilize the same components from sampleto sample, e.g., the same intake line, the same injector, the samepartitioner, the same reactor, and/or the same detection unit, withlittle or no sample-to-sample carryover between samples. For example, incertain embodiments, systems and methods are designed to flow a seriesof different samples through the components of the system where the samecomponent, e.g., the same intake line, the same injector, the samepartitioner, the same partition separator, and/or the same detector, isused for at least a first and a second different and consecutive samples(such as at least 2, 5, 10, 20, 50, 100, 200, 500, 1000, 5000, or 10,000samples), and where the system is configured so sample-to-samplecarryover from the first sample to the second sample is no more than 1%,or 0.1%, or 0.05%, or 0.01%, or 0.005%, or 0.001%, or 0.0005%, or0.0001%, or 0.00001%, or 0.000001%. Sample to sample carryover can bemeasured as described above for cross-contamination.

b) Within-sample alterations that affect processes occurring on theprocess side of the system can include any such alteration. Onealteration is coalescence of partitions within a sample (thoughcoalescence between samples can also occur).

c) Introduction of environmental material. A third source ofcross-contamination, as that term is used herein, includes introductionof material into a sample from the environment, e.g., dust, materialsfrom a user, and the like. While some of these materials are neutral interms of affecting the process side, others can have an effect. Forexample, particulate matter can clog one or more conduits. Particulatematter may also transit the system without introducing a clog but may befluorescent in one or more channels generating false positive results inthe data. Material from a user (e.g., from a sneeze or cough) cancontain nucleic acids which can affect, e.g., results of PCR processes.

Cross-contamination can occur in a number of ways, and systems andmethods provided herein can be designed to reduce or eliminate one ormore of these.

1) Barriers. If a container comprising a volume of fluid to be placedinto a process system, such as a container that contains a discretevolume of dispersed phase, e.g., a sample container, is used, where thecontainer, e.g., sample container, is open, material from one container,e.g., sample container can potentially move to another, e.g., if thesystem is jostled or similar event, or exterior material, e.g., dust,dirt, materials from the user, or the like, can enter the container. Inorder to prevent this, in certain embodiments, systems and methodsprovided herein can include one or more barriers between a discretevolume of fluid to be subject to intake into a system, and the exteriorenvironment, such as a seal over the top of the container in which thevolume of fluid is situated, or a non-sample-fluid layered over the top,or both. In certain cases, a holder comprising a plurality of containersof fluid to be placed into the process system, such as a plurality ofcontainers each of which contains a discrete volume of dispersed phase,e.g., a plurality of sample containers, such as a microtiter plate, isused, one or more barriers may be placed over the plurality ofcontainers of fluid. Such barriers can also be useful in preventingevaporation of a portion of the sample, which can affect the accuracy ofanalysis of the sample. Another barrier includes filters integrated intothe fluidic conduits delivering continuous phase or dispersed phase fromthe corresponding reservoirs. The filters are of an appropriate size toallow adequate flow rate of the material while maximizing the removal ofdebris. Another barrier can be a filter element that is positioned,e.g., at the intake end of an intake line. This can be as simple as anarrowing of the line at the end, to a dimension suitable to preventuptake of undesired particulate matter.

2) Cleaning exterior of intake conduit. If the same intake conduit isused to provide first and second samples to an intake system, materialfrom the first sample can adhere to the conduit and contaminate thesecond sample. Thus systems and methods provided herein include systemsand methods to remove dispersed phase, e.g., sample, adhering to anintake conduit. The systems and methods can be automated.

3) Surfaces with greater affinity for one fluid than another. If samplecomprises a fluid, e.g., a hydrophilic fluid such as water, then if oneor more surfaces that come in contact with the sample as it movesthrough the system (e.g., both intake and process system) have equal orgreater affinity for the fluid than for one or more other phases thatcome in in contact with the surface (e.g., than for a continuous phasewhen the sample is partitioned into a plurality of partitions ofdispersed phase in continuous phase), then part of the sample will tendto adhere to the surface, and can lag behind the rest of the sample,thus potentially coming in contact with, and contaminating, latersamples. Thus systems and methods provided herein include systems andmethods where surfaces that come in contact with a first fluid, e.g.,dispersed phase, such as sample, and at least one other fluid, e.g., atleast one other phase, such as continuous phase, have greater affinityfor the second fluid than for the first fluid, e.g., have greateraffinity for continuous phase than for dispersed phase. This can applyto the system as a whole, from intake side through process side, untilprocessed sample are beyond a detection zone, and/or to individual partsof a system, such as intake line, injector, partitioner, reactor (e.g.,thermal cycler), partition separation system (if used), and/or detector,as well as any connections between these. It will be appreciated that100% of the surface in contact with the first fluid, e.g., dispersedphase, such as sample, does not have to have the requisite affinities,so long as sufficient surface has requisite affinities so that a desiredlevel of cross-contamination, e.g., a desired maximum level, isachieved. Thus, in certain embodiments, at least 80, 90, 95, 98, 99,99.5, 99.9, 99.95, 99.99, 99.995, or 99.999% of surfaces that come incontact with first fluid, e.g., fluid of a sample, have greater affinityfor at least a second fluid that comes in contact with the surfaces, forexample, have less affinity for fluid of a sample, than for at least onesecond fluid that comes in contact with the surface, such as continuousphase; this applies to the system as a whole and independently to eachcomponent of the system, e.g., intake line, injector, partitioner,reactor, partition separator, detector, and/or conduits and connectionsbetween components.

4) Cleaning intake system. Systems and methods can be designed to cleanportions of an intake system that come in contact with dispersed phase,e.g., sample; in certain embodiments, this can be done while the intakesystem is isolated from a process system. For example, an intake systemcan be cleaned between samples so that no, or very little, of one sampleis carried over by the intake system into the process system with afollowing sample or samples, while leaving the process systemundisturbed. Thus, in a system where serial aliquots of dispersed phase,e.g., serial samples, are placed into the process system, in certainembodiments the intake system can be cleaned between aliquots ofdispersed phase, e.g., between samples, without disturbing the processsystem. Thus, in certain embodiments, provided herein are continuousflow serial emulsion systems that comprise an intake system and aprocess system, where the intake system can be isolated from the processsystem, e.g., for cleaning, such as between intake of a series ofaliquots of dispersed phase on the intake side, e.g., intake of samples.This can be accomplished in any suitable manner, such as describedfurther herein.

In certain embodiments, an injector is positioned between the intakesystem and the process system, where the injector is configured to movebetween being in fluid communication with the intake system and not influid communication with the process system and being in fluidcommunication with the process system and not in fluid communicationwith the intake system, e.g., so that the intake system and the processsystem are never in continuous fluid communication. In the configurationin which the injector is in fluid communication with the process systemand not in fluid communication with the intake system, the injector cantransport a portion or all of dispersed phase, e.g., a sample, into theprocess side, for example a portion or all of dispersed phase, e.g., asample, that is in a common conduit of the injector; while in theconfiguration in which the injector is in fluid communication with theintake system and not in fluid communication with the process system,the intake system, and the injector, can be cleaned between aliquots ofdispersed phase, e.g., between samples. Cleaning can include one or moreof purging and/or denaturing steps, and any other suitable steps, suchas described more fully herein.

5) Spacer fluid. Where serial aliquots of dispersed phase, e.g., serialsamples, are moved into a process system in a conduit as an emulsion ina continuous phase, axial dispersion may cause one aliquot, e.g., onesample, to overlap with another as they move through the system. One wayto minimize or eliminate overlap of successive aliquots of dispersedphase, e.g., successive samples, is to place a spacer fluid betweensuccessive aliquots, e.g., samples, preferably a spacer fluid that isimmiscible with dispersed phase in the aliquots, e.g., samples. Thus, incertain embodiments systems and methods provide for insertion of aspacer fluid between successive aliquots of dispersed phase, e.g.,successive samples, that enter a process system. Such a spacer fluid maybe provided, e.g., by an intake system, or other suitable system, beforeor after a partitioner; if before a partitioner, spacer fluid may bebroken into a plurality of partitions but, in general, has a compositionsuch that it reforms into a continuous plug. In embodiments where forexample, in systems where an injector is positioned between an intakesystem and a process system, the injector and the intake system can,e.g., transport an aliquot of dispersed phase, e.g., sample, into theprocess system, then the injector and intake system can inject a spacerfluid into the process system. The injector and intake system can,optionally, be cleaned, for example, between the injection of an aliquotof dispersed phase, e.g., sample and injection of the spacer fluid.

6) Reduction of flow disturbances. Flow in the system, e.g., in conduitsof the system, can be subject to disturbances that cause one aliquot ofdispersed phase, e.g., one sample, to be held up in the system andpotentially be available to contaminate one or more following aliquotsof dispersed phase, e.g., one or more following samples. Disturbances inflow can also encourage coalescence of partitions. Typically, systemsand methods provided herein are configured to cause laminar flow throughconduits of the system.

Connections Disturbances to flow can occur at connections betweencomponents of the system, if a smooth transition is not made between aconduit on one side of the connection and a conduit on the other side ofthe connection. An exemplary disturbance may include the creation ofmicroeddies at connections where a self is generated between twoconduits of different cross-sectional diameters. These microeddies canlead to sample hold up leading to partition transiting between samplesor introduce high shear force to partitions resulting in coalescence.Thus, in certain embodiments, connections between a first conduit and asecond conduit, are configured in such a way as to not disturb flow, orminimally disturb flow, from one side to the other, for example, theconnection can be made so that the cross-section of a first side of aconnection matches a cross-section of a second side of the connectionwhere the first and second sides are at a junction in the connection,and where second side is downstream of the first side, for example,matches within 80, 90, 95, 98, 99, 99.5, or 99.9% (e.g., cross-sectionsof first and second sides overlap to at least this degree), and/or wherelittle or no space (gap) occurs in the connection, e.g., the connectioncomprises no gap, or a gap that is less than 20, 15, 10, 5, 2, 1, 0.5,0.1, 0.01, 0.001, or 0.0001% the length of a characteristic dimension ofthe first conduit; for example, at a connection between a conduit of aninjector and a conduit leading from the injector, and/or at a connectionbetween a conduit (such as a conduit from the injector) and a conduit ofa partitioner, and/or at a connection between a conduit of a partitionerand a conduit exiting the partitioner (such as a conduit leading to areactor), and/or at a connection between a conduit (such as a conduitleading from a reactor) and a conduit of a partition separator, and/orat a connection between a conduit of a partition separator and a conduitexiting the partition separator (such as a conduit leading to adetector), and/or a connection between a conduit (such as a conduit froma partition separator, or from a reactor) and a conduit of a detector,and/or a connection between a conduit of a detector and an exit conduitfrom the detector (such as a conduit downstream from the detector).

Direction change Flow disturbance can also occur where a conduit changesdirection. If the direction change is too abrupt, depending on flow rateand cross-sectional area of the conduit and the like, an area ofturbulence or other disturbance will be created. Thus, in certainembodiments, the combination of flow rate, cross-sectional area, andradius of curvature of conduits in the system is such that shear forcesin the conduit are minimized. It will be appreciated that in certaincomponents, for example, in partitioners or partition separators, shearforces may deliberately be created, e.g., to create partitions, and suchare not included in this flow description. In certain embodiments,fluidic velocity can be, e.g., 0.15 mm/s-858 mm/s, such as 1-50 mm/s or5-10 mm/s.

7) Reducing buoyancy effects Buoyancy effects can also cause one aliquotof dispersed phase or portions thereof, to move in such a way as tooverlap with a second aliquot of dispersed phase or portions thereof,e.g., when an aliquot of dispersed phase, such as a sample, ispartitioned into a plurality of partitions and the partitions movethrough a conduit in a continuous phase, where the dispersed phase andcontinuous phase have properties such that one tends to be buoyant inthe other, e.g., dispersed phase tends to rise in continuous phase,buoyancy effects in the conduit can cause uneven flow of partitions, sothat partitions from one sample can overlap with those of anothersample. These effects can be minimized if the conduit comprising thepartitions is kept in a plane or nearly in a plane such that flow in theconduit is orthogonal to gravity, for example, within 45, 30, 20, 15,10, 5, 4, 3, 2, or 1 degree of a plane orthogonal to gravity. Inparticular, in certain embodiments, at least 80, 90, 95, 96, 97, 98, or99% of a portion of a conduit from the exit of a partitioner, or aconduit leading from the exit in the case where the exit is notorthogonal to gravity, to a separator for separating partitions fordetection, or, if such a separator is not used, to a detector, is within45, 30, 20, 15, 10, 5, 4, 3, 2, or 1 degree of a plane orthogonal togravity as measured from the axis of flow through the conduit.

8) Surfactants Systems and methods provided herein can include the useof surfactants. Surfactants can stabilize individual partitions incontinuous phase and can reduce coalescence of partitions. To stabilizeemulsions against coalescence, surfactants are used to lower theinterfacial tension and thus the Gibbs free energy, provide steric orelectrostatic repulsion, increase film drainage time, or increase thesurface elasticity. Emulsifiers mostly are amphiphilic moleculescomprising groups soluble in each of the two phases. When present in asingle solvent, either aqueous or oil, they form micellular structures.At the time of and for some period after, the micelles disperse andadsorb to an oil-water interface.

Surfactant added to the sampling side of the injector may preventdisruption of the dispersed phase packet after injection into theprocess side of the system before the dispersed phase packet issubdivided by the partitioner. Disruption of the dispersed phase packetprior to reaching the partitioner may result in sample hold up andpossible cross-contamination as well as reduce the consistency ofsubdivide partition sizes generated by the partitioner. Surfactant maybe introduced at one or more suitable points in the system, e.g., at theinjector and/or the partitioner, etc. One benefit to injectorintroduction is to minimize contamination due to prevention of aqueousphase adsorption to the conduit as well as stabilizing the sample packetbefore it reaches the partitioner so it doesn't fragment and leave apart of the sample packet in the conduit. Surfactant can be added to anysuitable concentration, such as 0.1-5%, 0.5-2%, or 0.5-1.5%, or0.8-1.2%, expressed as w/v, in general in the continuous phase as itenters a partitioner. In certain embodiments, the surfactant comprises afluorosurfactant; in certain embodiments the continuous phase comprisesa fluorinated oil and a fluorosurfactant.

Thus, systems and methods for serial flow emulsion reactions maycomprise use of a surfactant. The surfactant may stabilize droplets ofdispersed phases or continuous phases such that droplets do not coalescewhen in proximity.

In some instances, the surfactant is a fluorocarbon, a hydrocarbon, or asilicone. In some instances, the surfactant is a fluorosurfactant.

A volume of surfactant used may vary. In some instances, the volume ofsurfactant depends on a volume of the dispersed phase. In someinstances, the volume of the surfactant is a droplet of at least orabout 0.001 nanoliter (nL), 0.002 nL, 0.003 nL, 0.004 nL, 0.005 nL,0.006 nL, 0.007 nL, 0.008 nL, 0.009 nL, 0.01 nL, 0.02 nL, 0.03 nL, 0.04nL, 0.05 nL, 0.06 nL, 0.07 nL, 0.08 nL, 0.09 nL, 0.10 nL, 0.20 nL, 0.30nL, 0.40 nL, 0.50 nL, 0.60 nL, 0.70 nL, 0.80 nL, 0.90 nL, 1.0 nL, 2.0nL, 3.0 nL, 4.0 nL, 5.0 nL, or more than 5.0 nL. In some instances, thevolume of the surfactant is in a range of about 0.01 nL to about 1.5 nL.In some instances, a volume of the surfactant is at least or about 0.01nL, 0.02 nL, 0.03 nL, 0.04 nL, 0.05 nL, 0.06 nL, 0.07 nL, 0.08 nL, 0.09nL, 0.10 nL, 0.20 nL, 0.30 nL, 0.40 nL, 0.50 nL, 0.60 nL, 0.70 nL, 0.80nL, 0.90 nL, 1.0 nL, 2.0 nL, 3.0 nL, 4.0 nL, 5.0 nL, or more than 5.0nL. In some instances, the volume of the surfactant is in a range ofabout 0.1 nL to about 0.75 nL.

In certain embodiments, the emulsion, e.g. as it exits the partitioner,comprises surfactant at a level of less than 2, 1.7, 1.5, 1.4, 1.3, 1.2,1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.3, or 0.1% and/or at least 1.7,1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.3, 0.1 or0.05%, such as 0.5-2.0%, for example 0.2-1.5%, in some instances0.2-1.3%. In certain embodiments, dispersed phase, e.g. as it exits thepartitioner, comprises surfactant at a level of less than 2, 1.7, 1.5,1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.3, or 0.1% and/or atleast 1.7, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.3,0.1 or 0.05%, such as 0.2-2.0%, for example 0.2-1.5%, in some instances0.2-1.3%. However, other methods of expressing surfactant concentration,such as concentration in continuous phase entering a partitioner, mayalso be used, as described herein.

Surfactants and other components of dispersed phase, e.g., of an aqueoussample, as well as components of continuous phase, and other materialsuseful in systems and methods provided herein, are described further,below.

In certain embodiments, provided herein are systems and methods ofserial flow of emulsion, for example systems and methods comprising anintake system and a process system, such as described more fully herein,that, when used to process a series of samples, have levels ofcross-contamination between samples of less than 0.1%, for example, lessthan 0.01%, such as less than 0.005%. Thus, in certain embodiments,provided herein are systems and methods of serial flow of emulsion, forexample systems and methods comprising an intake system and a processsystem, that can include at least one of 1) a barrier between a discretevolume of fluid to be subject to intake into a system to provide serialflow of emulsions, and the exterior environment, such as a seal over thetop of the container in which the volume of fluid is situated; incertain cases a holder comprising a plurality of containers of fluid tobe placed into the process system, such as a plurality of containerseach of which contains a discrete volume of dispersed phase, e.g., aplurality of sample containers, such as a microtiter plate, and one ormore barriers positioned over the plurality of containers of fluid; 2) asystem or method to remove dispersed phase, e.g., sample, adhering to anexterior surface of an intake conduit; 3) surfaces that come in contactwith a first fluid, e.g., dispersed phase, such as sample, and at leastone other fluid, e.g., at least one other phase, such as continuousphase, having greater affinity for the second fluid than for the firstfluid, e.g., having greater affinity for continuous phase than fordispersed phase, e.g., at least 80, 90, 95, 98, 99, 99.5, 99.9, 99.95,99.99, 99.995, or 99.999% of surfaces that come in contact with firstfluid have greater affinity for at least a second fluid that comes incontact with the surfaces, for example, have greater affinity forcontinuous phase than for dispersed phase, e.g., sample; 4) an intakesystem for providing aliquots of dispersed phase, e.g., samples, to aprocess system, where the intake system can be cleaned between aliquotsof dispersed phase, e.g., between samples, without disturbing theprocess system; for example, where an injector is positioned between theintake system and the process system, where the injector is configuredto move between being in fluid communication with the intake system andnot in fluid communication with the process system and being in fluidcommunication with the process system and not in fluid communicationwith the intake system, but not both simultaneously, e.g., so that theintake system and the process system are never in continuous fluidcommunication and, in the configuration in which the injector is influid communication with the process system and not in fluidcommunication with the intake system, the injector can transport aportion or all of dispersed phase, e.g., a sample, into the processside, for example a portion or all of dispersed phase, e.g., a sample,that is in an injection chamber (common conduit) of the injector; whilein the configuration in which the injector is in fluid communicationwith the intake system and not in fluid communication with the processsystem, the intake system, and the injector, can be cleaned betweenaliquots of dispersed phase, e.g., between samples; 5) systems andmethods that provide for insertion of a spacer fluid between successivealiquots of dispersed phase, e.g., successive samples, that enter aprocess system; 6) connections between a first conduit and a secondconduit, where the first and second conduits are different, areconfigured in such a way as to not disturb flow, or minimally disturbflow, from one side to the other, for example, the connection can bemade so that the cross-section of a first side of a connection matches across-section of a second side of the connection where the first andsecond sides are at a junction in the connection, and where second sideis downstream of the first side, for example, matches within 80, 90, 95,98, 99, 99.5, or 99.9% (e.g., cross-sections of first and second sidesoverlap to at least this degree), and/or where little or no space (gap)occurs in the connection, e.g., the connection comprises no gap, or agap that is less than 20, 15, 10, 5, 2, 1, 0.5, 0.1, 0.01, 0.001, or0.0001% the length of a characteristic dimension of the first conduit;for example, at a connection between a conduit of an injector and aconduit leading from the injector, and/or at a connection between aconduit (such as a conduit from the injector) and a conduit of apartitioner, and/or at a connection between a conduit of a partitionerand a conduit exiting the partitioner (such as a conduit leading to areactor), and/or at a connection between a conduit (such as a conduitleading from a reactor) and a conduit of a partition separator, and/orat a connection between a conduit of a partition separator and a conduitexiting the partition separator (such as a conduit leading to adetector), and/or a connection between a conduit (such as a conduit froma partition separator, or from a reactor) and a conduit of a detector,and/or a connection between a conduit of a detector and an exit conduitfrom the detector (such as a conduit downstream from the detector); 7) acombination of flow rate, cross-sectional area, and radius of curvatureof conduits in the system is such that flow in the conduits is laminaror substantially laminar, 8) at least 80, 90, 95, 96, 97, 98, or 99% ofa portion of a conduit from an exit of a partitioner, or a conduitleading from the exit in a case where the exit is not orthogonal togravity, to a separator for separating partitions for detection, or, ifsuch a separator is not used, to a detector, is within 45, 30, 20, 15,10, 5, 4, 3, 2, or 1 degree of a plane orthogonal to gravity as measuredfrom the axis of flow through the conduit; and/or 9) surfactants, suchas one or more surfactants as described herein, e.g., at a level in acontinuous phase flowing into an inlet of partitioner of between 0.5 and2%, or between 0.5 and 1.5%, or between 0.8 and 1.2%. The systems andmethods can provide a level of cross contamination that is less than 20,10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.01, 0.05, 0.01, or 0.005%. Any orall of the preceding can be accomplished in a system or method thatutilizes the same components from sample to sample, e.g., the sameintake line, the same injector, the same partitioner, the same reactor,and/or the same detection unit. For example, in certain embodiments,systems and methods are designed to flow a series of different samplesthrough the components of the system where the same component, e.g., thesame intake line, the same injector, the same partitioner, the samepartition separator, and/or the same detector, is used for at least afirst and a second different and consecutive samples (such as at least2, 5, 10, 20, 50, 100, 200, 500, 1000, 5000, or 10,000 samples). Incertain embodiments, provided herein are systems and methods of serialflow of emulsion, for example systems and methods comprising an intakesystem and a process system, such as described more fully herein, that,when used to process a series of samples, have levels ofcross-contamination between samples of less than 0.1%, for example, lessthan 0.01%, such as less than 0.005%. Thus, in certain embodiments,provided herein are systems and methods of serial flow of emulsion, forexample systems and methods comprising an intake system and a processsystem, that can include at least two of 1)-9), above. In certainembodiments, provided herein are systems and methods of serial flow ofemulsion, for example systems and methods comprising an intake systemand a process system, such as described more fully herein, that, whenused to process a series of samples, have levels of cross-contaminationbetween samples of less than 0.1%, for example, less than 0.01%, such asless than 0.005%. Thus, in certain embodiments, provided herein aresystems and methods of serial flow of emulsion, for example systems andmethods comprising an intake system and a process system, that caninclude at least three of 1)-9), above. In certain embodiments, providedherein are systems and methods of serial flow of emulsion, for examplesystems and methods comprising an intake system and a process system,such as described more fully herein, that, when used to process a seriesof samples, have levels of cross-contamination between samples of lessthan 0.1%, for example, less than 0.01%, such as less than 0.005%. Thus,in certain embodiments, provided herein are systems and methods ofserial flow of emulsion, for example systems and methods comprising anintake system and a process system, that can include at least four of1)-9), above. In certain embodiments, provided herein are systems andmethods of serial flow of emulsion, for example systems and methodscomprising an intake system and a process system, such as described morefully herein, that, when used to process a series of samples, havelevels of cross-contamination between samples of less than 0.1%, forexample, less than 0.01%, such as less than 0.005%. Thus, in certainembodiments, provided herein are systems and methods of serial flow ofemulsion, for example systems and methods comprising an intake systemand a process system, that can include at least five of 1)-9), above. Incertain embodiments, provided herein are systems and methods of serialflow of emulsion, for example systems and methods comprising an intakesystem and a process system, such as described more fully herein, that,when used to process a series of samples, have levels ofcross-contamination between samples of less than 0.1%, for example, lessthan 0.01%, such as less than 0.005%. Thus, in certain embodiments,provided herein are systems and methods of serial flow of emulsion, forexample systems and methods comprising an intake system and a processsystem, that can include at least six of 1)-9), above. In certainembodiments, provided herein are systems and methods of serial flow ofemulsion, for example systems and methods comprising an intake systemand a process system, such as described more fully herein, that, whenused to process a series of samples, have levels of cross-contaminationbetween samples of less than 0.1%, for example, less than 0.01%, such asless than 0.005%. Thus, in certain embodiments, provided herein aresystems and methods of serial flow of emulsion, for example systems andmethods comprising an intake system and a process system, that caninclude at least seven of 1)-9), above. In certain embodiments, providedherein are systems and methods of serial flow of emulsion, for examplesystems and methods comprising an intake system and a process system,such as described more fully herein, that, when used to process a seriesof samples, have levels of cross-contamination between samples of lessthan 0.1%, for example, less than 0.01%, such as less than 0.005%. Thus,in certain embodiments, provided herein are systems and methods ofserial flow of emulsion, for example systems and methods comprising anintake system and a process system, that can include at least eight of1)-9), above. In certain embodiments, provided herein are systems andmethods of serial flow of emulsion, for example systems and methodscomprising an intake system and a process system, such as described morefully herein, that, when used to process a series of samples, havelevels of cross-contamination between samples of less than 0.1%, forexample, less than 0.01%, such as less than 0.005%. Thus, in certainembodiments, provided herein are systems and methods of serial flow ofemulsion, for example systems and methods comprising an intake systemand a process system, that can include all of 1)-9), above.

Sample processing. Sample processing applications including cell lysis,cell growth, ligation, digestions, nucleic acid assembly reactions,nucleic acid editing, nucleic acid modification, or sample analysisincluding the detection of nucleic acids, proteins, and microbialorganisms using reactions include but are not limited to RNAtranscription, hybridization chain reaction (HCR), nicking chainreaction, loop-mediated isothermal amplification (LAMP), stranddisplacement amplification (SDA), helicase-dependent amplification(HDA), nicking enzyme amplification reaction (NEAR), nucleic acid melttemperature analysis, protein detection, protein melt temperatureanalysis, small molecule detection, microbial growth rate testing,antibiotic resistance testing, microbial small molecule production,molecule-molecule interaction studies. These reactions may occur at onemore fixed temperature also known as isothermal reactions or may undergotemperature cycling to promote reaction progress. Many reactions can becombined in the same partitions For example the quantification of mRNAand protein from the same sample, or the same cell, can generatebiologically relevant information only available when the reactions arecontained within the same partition.

RNA transcription reactions are isothermal reactions carried out by RNApolymerase enzymes. The polymerases bind to DNA sequences bearingpromoter sequences to initiate the production of RNA in a templatedirected reaction. Monitoring the production of RNA allows this reactionto be used for the detection of the DNA sequences bearing particularpromoters.

In an example of an RNA transcription reaction the aqueous reactionphase would be prepared to consist of appropriate buffer, for instanceTris-HCl pH 8.5, nucleotide triphosphates, magnesium chloride, sodiumchloride, dithiothreitol, reporter system, for example an RNA bindingdye, fluorescent nucleotide triphosphate derivative, or a hybridizationprobe like a molecular beacon, and an RNA polymerase. The aqueousreaction phase is combined with an aqueous sample to be tested for thepresence of particular DNA sequences. The combined sample is then readyfor injection into the process stream of the instrument. After injectionthe sample is partitioned so that sample partitions can be incubate at afixed temperature, for instance 37 C, for fixed period of time, forexample 1 min, 5 min, 15 min, 30 min, 45 min, 1 hr, 2 hr, 4 hr, 8 hr.The intensity of the reporter system in the partition is theninterrogated in order to identify the presence of the DNA sequences ofinterest.

HCR is an isothermal nucleic acid detection method that utilizes aseries of metastable hairpins that upon binding to a target nucleic acidexponentially unfold. In HCR, a target nucleic acid is added to mixtureof two or more metastable hairpin molecules. Upon binding the targetnucleic acid sequence, the first of said hairpins opens exposing aregion complementary to the second of said hairpins. This process, inturn, exposes a single-stranded region identical to the first of saidhairpins. The resulting chain reaction leads to the formation of anicked double helix that grows until the hairpin supply is exhausted.HCR is capable of detecting both DNA and RNA molecules.

LAMP is an isothermal nucleic acid amplification reaction that employseither two or three sets of primers and a polymerase with high stranddisplacement activity in addition to a replication activity. Typically,4 different primers are used to identify 6 distinct regions on thetarget gene, which adds highly to the specificity. An additional pair of“loop primers” can further accelerate the reaction. Due to the specificnature of the action of these primers, the amount of DNA produced inLAMP is considerably higher than PCR based amplification. LAMP iscapable of detecting both DNA and RNA molecules.

SDA is an isothermal DNA amplification reaction that relies on astrand-displacing DNA polymerase, typically Bst DNA Polymerase, LargeFragment or Klenow Fragment (3′-5′ exo-), to initiate at nicks createdby a strand-limited restriction endonuclease or nicking enzyme at a sitecontained in a primer. The nicking site is regenerated with eachpolymerase displacement step, resulting in exponential amplification.SDA is capable of detecting both DNA and RNA molecules.

HDA employs the double-stranded DNA unwinding activity of a helicase toseparate strands, enabling primer annealing and extension by astrand-displacing DNA polymerase. Since an enzyme replaces thedenaturing step used in traditional PCR, HDA reactions proceed at asingle temperature and result in logarithmic amplification of the targetDNA.

NEAR employs a strand-displacing DNA polymerase initiating at a nickcreated by a nicking enzyme, rapidly producing many short nucleic acidsfrom the target sequence. This process is extremely rapid and sensitive,enabling detection of small target amounts in minutes. The nickingenzyme and polymerase are precisely matched to function at the sametemperature removing the need for thermal cycling. NEAR is capable ofdetecting both DNA and RNA molecules.

An exemplary NEAR reaction is, prepare aqueous phase consisting ofappropriate buffer like tris pH 8.5, deoxynucleotide triphosphates,magnesium chloride, sodium chloride, BSA, a suitable nicking enzymedescribed below, and a suitable polymerase described below. Combineaqueous phase with nucleic acid to be tested. Inject sample into processside. Partition sample. Incubate sample at desired temperature forpolymerase and nickase function for fixed period of time, for example 1min, 5 min, 15 min, 30 min, 45 min, 1 hr, 2 hr, 4 hr, 8 hr. Analyze DNAproduction with suitable reporter including DNA binding dyes,fluorescent nucleotide triphosphate derivatives, hybridization probeslike molecular beacons. Nicking specific hybridization probes may beused. Nicking probes might look like a fluorophore and quencher linkedto a oligonucleotide between 5-20 nt apart with a nickase recognitionsequence internal to the oligonucleotide. In this example the probebinds to single stranded DNA produced by the polymerase, uponcomplementation, the probe generates a double stranded nicking siterecognizable by the nicking enzyme. Upon nicking the probe, the oligo iscleaved separating the fluorophore and quencher allowing for theproduction of a detectable signal. Quantify the amount of fluorescencewith the detector assembly.

RT-PCR is a method of RNA detection that relies on first converting theRNA to its complementary DNA form called cDNA and then amplifying thecDNA by PCR.

An exemplary RT-PCR reaction is, prepare aqueous phase consisting ofappropriate buffer like tris pH 8.5, deoxynucleotide triphosphates,magnesium chloride, sodium chloride, BSA, a suitable reversetranscriptase described below, and a suitable polymerase describedbelow, primers, and detection reagents. Combine aqueous phase withnucleic acid to be tested. Inject sample into process side. Partitionsample. Incubate sample at desired temperature for polymerase functionfor fixed period of time, for example 1 min, 5 min, 15 min, 30 min, 45min, 1 hr, 2 hr, 4 hr, 8 hr. Thermal cycle the reaction. Analyze DNAproduction with suitable reporter including DNA binding dyes,fluorescent deoxynucleotide triphosphate derivatives, hydrolysis probeslike taq man probes. Quantify the amount of fluorescence with thedetector assembly.

Polymerases useful in the methods described herein are capable ofcatalyzing the incorporation of nucleotides to extend a 3′ hydroxylterminus of an oligonucleotide bound to a target nucleic acid molecule.Such polymerases include those capable of amplification and/or stranddisplacement. The polymerase may bear or lack 5′-3′ exonucleaseactivity. In other embodiments, a polymerase also has reversetranscriptase activity (e.g., Bst (large fragment), Therminator,Therminator II). Exemplary polymerases include but are not limited toBST (large fragment), DNA polymerase I (E. coli), DNA polymerase I,Large (Klenow) fragment, Klenow fragment (3′-5′ exo-), T4 DNApolymerase, T7 DNA polymerase, Deep VentR. (exo-) DNA Polymerase, DeepVentR DNA Polymerase, DyNAzyme, High-Fidelity DNA Polymerase,Therminator, Therminator II DNA Polymerase, AmpliTherm DNA Polymerase,Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tgo DNApolymerase, SP6 DNA polymerase, Thr DNA polymerase. The followingnon-limiting examples of Reverse Transcriptases (RT) can be used in thereactions of the present method to improve performance when detecting anRNA sequence: OmniScript, SensiScript, MonsterScript, Transcriptor, HIVRT, SuperScript III, ThermoScript, Thermo-X, ImProm II. The followingnon-limited examples of RNA polymerases include but are not limited toT3, T7, SP6, E. coli RNA pol, RNA pol II, and mtRNA pol.

A nicking enzyme binds double-stranded DNA and cleaves one strand of adouble-stranded duplex. The nicking enzyme may cleave either upstream ordownstream of the binding site or nicking enzyme recognition site. Inexemplary embodiments, the reaction comprises the use of a nickingenzyme that cleaves or nicks downstream of the binding site such thatthe product sequence does not contain the nicking site. Using an enzymethat cleaves downstream of the binding site allows the polymerase tomore easily extend without having to displace the nicking enzyme.Ideally, the nicking enzyme is functional under the same reactionconditions as the polymerase. Exemplary nicking include, but are notlimited to, Nt.BspQI(NEB), Nb.BbvCI(NEB), Nb.BsmI(NEB), Nb.BsrDI(NEB),Nb.BtsI(NEB), Nt.AlwI(NEB), Nt.BbvCI(NEB), Nt.BstNBI(NEB),Nt.CviPII(NEB), Nb.Bpu10I(Fermantas), and Nt.Bpu10I(Fermentas).

Fluorogenic substrates, a nonfluorescence material that when acted uponby an enzyme converts to a fluorescent state, may be used to identifythe presence of a particular protein with a partition. Fluorogenicsubstrates have been developed for the detection and characterization ofa wide array of enzyme classes.

An example of small molecule detection includes the detection ofbacterial endotoxin using a factor C assay. Gram negative bacterialendotoxin is a biological pyrogen that causes fever when introducedintravenously. The endotoxin, also known as lipopolysaccharide (LPS), isfound in the outer membrane of Gram-negative bacteria. DuringGram-negative sepsis, endotoxin stimulates host macrophages to releaseinflammatory cytokines. However, excessive inflammation causes multipleorgan failure and death. Endotoxins, which are ubiquitous pathogenicmolecules, are a bane to the pharmaceutical industry and healthcarecommunity. Thus early and sensitive detection of endotoxin is crucial toprevent endotoxaemia. The gold standard for LPS detection is the limulusamebocyte lysate (LAL) test and has been widely used for ˜30 years forthe detection of endotoxin in the quality assurance of injectable drugsand medical devices. The LAL constitutes a cascade of serine proteaseswhich are triggered by trace levels of endotoxin, culminating in a gelclot at the end of the reaction. The Factor C, which normally exists asa zymogen, is the primer of this coagulation cascade. In vivo, Factor Cis the perfect biosensor, which alerts the horseshoe crab of thepresence of a Gram-negative invader. The hemostatic end-point entrapsthe invader, killing it and limiting further infection. However, as anin vitro endotoxin detection tool, variations in the sensitivity andspecificity of LAL to endotoxin, and the dwindling supply of horseshoecrabs are posing increasing challenges to the biotechnology industry.Therefore, methods for the miniaturization and digitization of saidassay has the potential to lower the need for LAL as well as increaseits sensitivity.

Microbial growth studies using various metabolic sources may beperformed. Bacteria may be encapsulated in droplets with a specificmedium source at a single occupancy. The bacteria are incubated at aconstant temperature and then measured after a fixed period of time.Those bacteria capable of metabolizing the medium constituents will havea higher fitness and growth rate than bacteria incapable of metabolizingsaid medium. Antibiotic susceptibility studies may be performed in asimilar way. In these cases, bacteria may be encapsulated in thepresence and absence of specific antibiotics. Those bacteria susceptibleand those resistant will demonstrate slower or higher growth ratesrespectively. In these cases, bacteria may be monitored using dropletimaging and image processing, cell-specific stains and eitherfluorescence or absorption measurements, or be genetically modified toproduce datable reporter phenotypes including but not limited tofluorescent or colored proteins, reporter enzymes, etc.

Other specific examples include but are not limited to environmental DNAanalysis, forensic sample analysis, agricultural sample analysisincluding GMO and pathogen detection, detection of RNA and DNAmethylation patterns, detection of DNA damage, copy number variation,gene rearrangement analysis, splicing variants, DNA and RNA structuralrearrangements, SNP detection, antibody testing and/or identification,next generation sequencing library absolute quantification, viral loadquantification, telomere length testing, protein:nucleic acidcorrelations, gene editing efficiency, enzyme quantification.

Materials Used in Systems and Methods Provided Herein.

Dyes (fluorescent molecules). In certain embodiments, a fluorescentmoiety, such as a molecule may be used (fluorophores). Any suitablefluorescent moiety may be used. Examples of dyes include Eva Green, SYBRgreen I, SYBR green II, SYBR gold, ethidium bromide, methylene blue,Pyronin Y, DAPI, acridine orange, Blue View or phycoerythrin. A widevariety of reactive fluorescent probes can also be used. The fluorophorecan be an aromatic or heteroaromatic compound. The fluorophore can be,for example, a pyrene, anthracene, naphthalene, acridine, stilbene,benzoxaazole, indole, benzindole, oxazole, thiazole, benzothiazole,canine, carbocyanine, salicylate, anthranilate, xanthenes dye, coumarin.Exemplary xanthene dyes include, e.g., fluorescein and rhodamine dyes.Fluorescein and rhodamine dyes include, but are not limited to6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N;N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX).Suitable fluorescent probes also include the naphthylamine dyes thathave an amino group in the alpha or beta position. For example,naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonate,1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalenesulfonate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Exemplary coumarins include, e.g., 3-phenyl-7-isocyanatocoumarin;acridines, such as 9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl) maleimide; cyanines, such as, e.g.,indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5),indodicarbocyanine 5.5 (Cy5.5),3-(-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA); 1H, 5H,11H, 15H-Xantheno[2,3, 4-ij: 5,6, 7-i′j′]diquinolizin-18-ium, 9-[2 (or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4 (or2)-sulfophenyl]-2,3, 6,7, 12,13, 16,17-octahydro-inner salt (TR or TexasRed); or BODIPY™ dyes.

Methods to reduce partition coalescence include the use of fluorinatedoils, surfactants in either the continuous or discontinuous phase, usingdefined conduit cross-sectional areas, limiting any abrupt changes inconduit cross-sectional area, maintaining partition fluidic velocitieswithin a defined range within the conduits of the system, grounding thesystem to eliminate significant voltage potentials, includingdissipation of static charges, limiting the aggregation of partitionsthat can occur due to buoyant forces.

Surfactants demonstrate utility in both the injector and process sidesof the system. On the injector side, surfactants may stabilize dispersedphase packets upon injector into the process side. This will preventdisruption of the dispersed phase packet prior to reaching thepartitioner. Disruption of the dispersed phase prior to partitioning mayresult in sample hold up leading to cross-contamination, and/or willresult in higher variance is subdivided partition size upon partitioningat the partitioner.

In one embodiment, a single surfactant type is present in both injectionand process streams. In a second embodiment, a single surfactant ispresent only in the process stream. In a third embodiment two differentsurfactant types are used in the partitioner and injector streams butthe surfactants are at the same concentration. In a fourth embodimentthe relative concentrations of surfactants between the two streams isdifferent. In a fifth embodiment one or more surfactant types are usedin the injection stream and one or more surfactant type is used in theprocess stream.

In certain embodiments, a fluorinated oil is used, e.g., as continuousphase and/or for other fluid components as described further herein.Fluorinated oils may comprise(3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyle-hexane),methyl nonafluorobutyl ether, methyl nonafluoroisobutyl ether, ethylnonafluoroisobutyl ether, ethyl nonofluorobutul ether, (pentane,1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl-)),isopropyl alcohol, (1,2-trans-dichloroethylene),(butane,1,1,1,2,2,3,3,4,4-nonafluoro-4-methoxy-),(1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl)-3-pentanone),(furan,2,3,3,4,4-pentafluorotetrahydro-5-methoxy-2,5-bis[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]-),perfluoro compounds comprising between 5 and 18 carbon atoms,polychlorotrifluoroethylene, (2,2,2-trifluoroethanol), Novec 8200™,Novec 71DE™, Novec 7100™, Novec 7200DL™, Novec 7300DL™, Novec 71IPA™,Novec 72FL™, Novec 7500™, Novec 71DA™, Novec 7100DL™, Novec 7000™, Novec7200™, Novec 7300™, Novec 72DA™, Novec 72DE™, Novec 649™, Novec 73DE™,Novec 7700™, Novec 612™, FC-40™, FC-43™, FC-70™, FC-72™, FC-770™,FC-3283™, FC-3284™, PF-5056™, PF5058™, Halocarbon 0.8™, Halocarbon 1.8™,Halocarbon 4.2™, Halocarbon 6.3™, Halocarbon 27™, Halocarbon 56™,Halocarbon 95™, Halocarbon 200™, Halocarbon 400™, Halocarbon 700™,Halocarbon 1000N™, Uniflor 4622R™, Uniflor 8172™, Uniflor 8472CP™,Uniflor 8512S™, Uniflor 8731™, Uniflor 8917™, Uniflor 8951™, TRIFLUNOX3005™, TRIFLUNOX 3007™, TRIFLUNOX 3015™, TRIFLUNOX 3032™, TRIFLUNOX3068™, TRIFLUNOX 3150™, TRIFLUNOX 3220™, or TRIFLUNOX 3460™.

Components as described herein may comprise a surfactant; e.g., incertain embodiments a surfactant is in a continuous phase, but it willbe appreciated that surfactant can be added to the system at anysuitable point and in any suitable fluid. In certain embodiments, asurfactant is a fluorinated surfactant. In certain embodiments,fluorosurfactants comprise an oligoethylene glycol, TRIS, orpolyethylene glycol moiety. In certain embodiments, fluorosurfactantscomprise a fluorocarbon and/or chlorofluorocarbon moiety. In someembodiments, fluorosurfactants have head and tail moieties linked byether, amide, or carbamide bonds. In a preferred embodiment,fluorosurfactants have a polyethylene glycol moiety linked to afluorocarbon moiety through a carbamide, ether, or amide bond.Fluorinated surfactants include but are not limited to Picosurf-1, RanFS-008, FC-4430, FC-4432, FC-4434.

In certain embodiments, a fluorinated oil is used with afluorosurfactant.

In certain embodiments, the fluorinated oil comprises(3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyle-hexane),(furan,2,3,3,4,4-pentafluorotetrahydro-5-methoxy-2,5-bis[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]-),and/or perfluoro compounds comprising between 5 and 18 carbon atoms andthe fluorosurfactant comprises a polyethylene moiety linked to afluorocarbon moiety with a carbamide, amide, or ether bond.Fluorosurfactant can have a concentration between 0.01% w/v to 5% w/v inthe fluorinated oil. In certain embodiments, fluorosurfactantconcentration ranges from 0.5% to 2% w/v, such as 0.5-1.5%. In generalherein, surfactant concentrations are expressed as a percentage ofsurfactant in continuous phase, e.g., the percentage of surfactant inthe continuous phase as it is flowed into a partitioner to producepartitions of dispersed phase.

A dispersed phase, e.g., an aqueous phase may contain one or morebuffering components included in but not limited to the following list:1,3-Bis[tris(hydroxymethyl)-methylamino]propane (Bis-Tris-Propane),1,4-Piperazinediethanesulfonic acid (PIPES),2-(Cyclohexylamino)ethanesulfonic acid (CHES),2-(N-Morpholino)ethanesulfonic acid (MES),2-[(2-Hydroxy-1,1-bis[hydroxymethyl]ethyl)amino]ethanesulfonic acid(TES), 2-Amino-2-methyl-1-propanol (AMP),2-Amino-2-methyl-1,3-propanediol (AMPD), 2-Aminoethanesulfonic acid(AES), 2,2-Bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol (Bis-Tris),3-([1,1-Dimethyl-2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid(AMPSO), 3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS),3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),3-(N-Morpholino)propanesulfonic acid (MOPS),3-(N-Morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO),3-(N-tris[Hydroxymethyl]methylamino)-2-hydroxypropanesulfonic acid(TAPSO), 3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid(DIPSO), 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS),4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES),4-(Cyclohexylamino)-1-butanesulfonic acid (CABS),4-(N-Morpholino)butanesulfonic acid (MOBS), Acetic acid, Ammonia, Boricacid, Cacodylic acid, Carbonate-Bicarbonate, Carbonic acid,Citrate-dextrose, Citrate-phosphate-dextrose, Citric acid, Diglycine,Dimethylarsinic acid, Ethanolaminie, Ethylenediaminetetraacetic acid(EDTA), Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraaceticacid (EGTA), Formic acid, Glycine, Glycylglycine, Hydroxyacetic acid,Imidazole, Lactic acid, Malic acid, Maleic acid,N-(2-Acetamido)-2-aminoethanesulfonic acid,N-(Carbamoylmethyl)-2-aminoethanesulfonic acid (ACES),N-(2-Acetamido)-2-iminodiacetic acid (ADA),N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS),N-(2-Hydroxyethyl)-piperazine-N′-(2-hydroxypropanesulfonic acid)(HEPPSO), N-[Tris(hydroxymethyl)methyl]glycine (Tricine),N-tris(Hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),N,N-Bis(2-hydroxyethyl)glycine (Bicine), Phosphoric acid,Piperazine-1,4-bis(2-hydroxypropanesulfonic acid) (POPSO),Pyrophosphoric acid, Succinic acid, Tetraboric acid, Tricinie,Triethylammonium acetate, Triethylammonium bicarbonate, Triethylammoniumphosphate, Triethanolamine (TEA), Tris-acetate, Tris-acetate-EDTA,Tris-borate, Tris-borate-EDTA, Tris-EDTA, Tris-Glycine, Tris-Tricine,tris(hydroxymethyl)aminomethan (Tris).

A dispersed phase, e.g., an aqueous phase may contain one or moreprotease inhibitor included in but not limited by the following listthat may target aspartic, cysteine, metallo-, serine, threonine, andtrypsin proteases: Alpha-2-Macroglobulin, Antipain, Aprotinin,Benzamidine, Bestatin, Calpain inhibitor I and II, Chymostatin, E-64,Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid(EGTA), Ethylenediaminetetraacetic acid (EDTA), Leupeptin(N-acetyl-L-leucyl-L-leucyl-L-argininal), Pefabloc SC, Pepstatin,Phenylmethylsulfonyl fluoride (PMSF), Tosyl phenylalanyl chloromethylketone (TLCK), Trypsin inhibitors.

A dispersed phase, e.g., an aqueous phase may contain one or moreantimicrobial agent included in but not limited by the following list:Actinomycin D, Ampicillin, Anhydrotetracycline, Apramycin, Asinomycin,Azidothymidine, Azithromycin, Blasticidin, Bleocin, Carbenicillin,Cefazolin, Cefotaxime, Cefoxitin, Ceftazidime, Ceftriaxone, Cefuroxime,Cetrimide, Chloramphenicol, Ciprofloxacin, Clindamycin, Cotrimoxazole,Coumermycin, Cycloheximide, Cycloserine, Erthromycin, Erythromycin,Fungizone, Geneticin, Gentamycin, Hygromycin, Kanamycin, Kasugamycin,Levofloxacin, Linezolid, Mycophenolic Acid, Nafcillin, Nalidixic Acid,Neomycin, Novobiocin, Nystatin, Oxacillin, Oxolinic Acid, Penicillin,Pipemidic Acid, Polymyxin B, Puromycin, Rifampcin, Sodium azide,Spectinomycin, Streptomycin, Tetracycline, Thimerosal, Thiostrepton,Ticarcillin, Tobramycin, Triclosan, Vancomycin, Zeocin.

A dispersed phase, e.g. an aqueous phase may contain one or morecrowding agent included in but not limited by the following list:1,2-propanediol, Carboxymethyl cellulose, Ethylene glycol, Glycerol, PEG200, PEG 300, PEG 400, PEG 600, PEG 1000, PEG 1300, PEG 1600, PEG 1450,PEG 1500, PEG 2000, PEG 3000, PEG 2050, PEG 3350, PEG 4000, PEG 4600,PEG 6000, PEG 8000, PEG 10000, PEG 12000, PEG 20000, PEG 35000, PEG40000, PEG 108000, PEG 218000, PEG 510000, PEG 90M, Polysucrose,Polyvinyl alcohol, Polyvinylpyroolidone, Propylene glycol.

A dispersed phase, e.g., an aqueous phase may contain one or moredetergent included in but not limited by the following list:1-Octanesulfonic acid, 1-Oleoyl-rac-glycerol, 2-Cyclohexylethylβ-D-maltoside, 3-(1-Pyridinio)-1-propanesulfonate,3-(4-tert-Butyl-1-pyridinio)-1-propanesulfonate,3-(Benzyldimethylammonio)propanesulfonate,3-(Decyldimethylammonio)-propane-sulfonate inner salt zwitterionicdetergent, 3-(N,N-Dimethylmyristylammonio)propanesulfonate,3-(N,N-Dimethyloctadecylammonio)propanesulfonate,3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt,3-(N,N-Dimethylpalmitylammonio)propanesulfonate,3-[N,N-Dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate,4-Dodecylbenzenesulfonic acid, 4-Nonylphenyl-polyethylene glycol,5-Cyclohexylpentyl β-D-maltoside, 6-Cyclohexylhexyl β-D-maltoside,Alkyltrimethylammonium bromide, Amprolium hydrochloride, APO-10, APO-12,ASB-14, ASB-16, ASB-C80, Benzalkonium chloride, Benzethonium chloride,Benzethonium hydroxide, Benzyldimethyldodecylammonium chloride,Benzyldimethylhexadecylammonium chloride, Benzyldodecyldimethylammoniumbromide, Bile salts, BRIJ® 35 Detergent, Brij® 58, Brij® L23, Brij® L4,Brij® O10, BRIJ® O20, C₁₂E₈, C7BzO, Caprolyl sulfobetaine,Cetylpyridinium chloride, CHAPS, CHAPSO, Chenodeoxycholic acid, Cholicacid, Cremophor EL®, DDMAB, Decaethylene glycol mono-dodecyl ether,Decyl β-D-glucopyranoside, Decyl β-D-maltopyranoside,Decyl-β-D-1-thiomaltopyranoside, Decyl-β-D-maltoside, Deoxycholic acid,DGEA, Dicyclohexyl sulfosuccinate, Diethylene glycol, diethylene glycoloctadecyl ether, Digitonin, Digitoxigenin, Dihexadecyl phosphate,Dihexyl sulfosuccinate, Dimethyldioctadecylammonium bromide,Dimethylethylammoniumpropane sulfonate, Docusate sodium,Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium bromide,ELUGENT™ Detergent, EMPIGEN® BB Detergent, Ethanesulfonic acid, Ethyleneglycol monododecyl ether, Ethylene glycol monohexadecyl ether, Ethyleneglycol monohexyl ether, Ethylhexadecyldimethylammonium bromide, FC-4430,FC-4432, FC-4434, Genapol® C-100, Genapol® X-080, Genapol® X-100,Girard's reagent T, Glucopone 600 CS, Glycocholic acid, HECAMEG®,Hexadecyl(2-hydroxyethyl)dimethylammonium dihydrogen phosphate,Hexadecylpyridinium bromide, Hexadecylpyridinium chloride,Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium chloride,Hexadecyltrimethylammonium p-toluenesulfonate, Hexaethylene glycolmonododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethyleneglycol monotetradecyl ether, Hexyl β-D-glucopyranoside, IGEPAL® CA-630,IGEPAL® CA-720, Imbentin AGS/35, Isopropyl β-D-1-thiogalactopyranoside,Kolliphor® EL, L-α-Lysophosphatidylcholine, Lithium3,5-diiodosalicylate, Lithium dodecyl sulfate, Lugol, Lutrol® OP 2000,Luviquat™ FC 370, Luviquat™ FC 550, Luviquat™ HOLD, Luviquat™ Mono LS,Methoxypolyethylene glycol 350, Methyl6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside, Methylbenzethoniumchloride, Miltefosine, Myristyltrimethylammonium bromide,N-Decanoyl-N-methylglucamine, N-Decanoylsucrose,N-Decyl-β-D-maltopyranoside, N-Dodecanoylsucrose, N-Dodecylβ-D-glucopyranoside, N-Dodecyl β-D-maltoside,N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,N-Dodecyl-β-D-glucopyranoside, N-Dodecyl-β-D-maltoside, N-Heptylβ-D-glucopyranoside, N-Heptyl β-D-thioglucopyranoside, N-Hexadecylβ-D-maltoside, N-Lauroyl-L-alanine, N-Lauroylsarcosine,N-Nonanoyl-N-methylglucamine, N-Nonyl-β-D-glucopyranoside,N-Octanoyl-N-methylglucamine ≥97%, N-Octanoylsucrose, N-Octylβ-D-maltoside, N-Octyl-β-D-glucopyranoside,N-Octyl-β-D-thioglucopyranoside,N,N-Bis[3-(D-gluconamido)propyl]deoxycholamide, N,N-DimethyldecylamineN-oxide, N,N-Dimethyldodecylamine N-oxide, NDSB 211, NDSB-195, NDSB-201,NDSB-256, Niaproof® 4, Nonaethylene glycol monododecyl ether, Nonidet™ P40, Nonyl β-D-glucopyranoside, Nonyl β-D-maltoside,Nonyl-β-D-1-thiomaltoside, Nonylphenyl-polyethyleneglycol acetate,O-(Decylphosphoryl)choline, Octaethylene glycol monodecyl ether,Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecylether, Octaethylene glycol monooctadecyl ether, Octylβ-D-1-thioglucopyranoside, Octyl β-D-glucopyranoside,Octyl-α/β-glucoside, Octyl-β-D-glucopyranoside, Pentaethylene glycolmonodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethyleneglycol monohexyl ether, Pentaethylene glycol monooctyl ether, Pluronic®F-127, Pluronic® F-68, Poloxamer 188, Poloxamer 407, Poly(maleicanhydride-alt-1-decene), 3-(dimethylamino)-1-propylamine, Poly(maleicanhydride-alt-1-tetradecene), 3-(dimethylamino)-1-propylamine,Polyoxyethylene (10) tridecyl ether, Polyoxyethylene (20) sorbitanmonolaurate, Polyoxyethylene (40) stearate, Polysorbate 20, Polysorbate60, Polysorbate 80, Saponin, SB 3-10, SB 3-14, Sodium 1-butanesulfonate,Sodium 1-decanesulfonate, Sodium 1-heptanesulfonate, Sodium1-hexanesulfonate, Sodium 1-nonanesulfonate, Sodium 1-octanesulfonate,Sodium 1-pentanesulfonate, Sodium 1-propanesulfonate, Sodium2-ethylhexyl sulfate, Sodium 2,3-dimercaptopropanesulfonate, Sodiumchenodeoxycholate, Sodium choleate, Sodium cholesteryl sulfate, Sodiumdeoxycholate, Sodium dodecyl sulfate, Sodium glycochenodeoxycholate,Sodium glycocholate hydrate, Sodium glycodeoxycholate, Sodiumhexanesulfonate, Sodium octanoate, Sodium octyl sulfate, Sodiumpentanesulfonate, Sodium taurochenodeoxycholate, Sodium taurocholatehydrate, Sodium taurodeoxycholate hydrate, Sodium taurohyodeoxycholatehydrate, Sodium taurolithocholate, Sodium tauroursodeoxycholate,SODOSIL™ RM 003, SODOSIL™ RM 01, Span® 20, Span® 60, Span® 65, Span® 80,Span® 85, Sucrose monodecanoate, Sucrose monolaurate, Surfactin,Synperonic® F 108, Synperonic® PE P105, Synperonic® PE/P84, Taurocholicacid, Taurolithocholic acid 3-sulfate, Teepol™ 610 S, TERGITOL™ MINFOAM, TERGITOL™ TMN 10, TERGITOL™ TMN 6, TERGITOL™ Type 15-S-5,TERGITOL™ Type 15-S-7, TERGITOL™ Type 15-S-9, TERGITOL™ Type NP-10,TERGITOL™ Type NP-9, Tetradecyl-β-D-maltoside, Tetraethylene glycolmonododecyl ether, Tetraethylene glycol monooctadecyl ether,Tetraethylene glycol monooctyl ether, Tetraglycol, Tetraheptylammoniumbromide, Tetrakis(decyl)ammonium bromide, Tetramethylammonium hydroxide,Thesit®, Tri-C8-10-alkylmethylammonium chloride, Tridecyl β-D-maltoside,Tridodecylmethylammonium chloride, Triethylene glycol monodecyl,Triethylene glycol monomethyl ether, Triton™ N-57, Triton™ N-60, Triton™QS-15, Triton™ X-100, Triton™ X-102, Triton™ X-114, Triton™ X-114,Triton™ X-165, Triton™ X-305, Triton™ X-405, Triton™ X-45, Turkey redoil, Tween® 20, Tween® 40, Tween® 60, Tween® 65, Tween® 80, Tween® 85,Tyloxapol, Undecyl β-D-maltoside, Ursodeoxycholic acid, ZWITTERGENT®3-08, ZWITTERGENT® 3-10, ZWITTERGENT® 3-12, ZWITTERGENT® 3-14,ZWITTERGENT® 3-16.

A dispersed phase, e.g., an aqueous phase, may comprise one or morenucleotide or derivatives of said nucleotides included in but notlimited by the following list: 5-Fluoroorotic Acid (5-FOA), Adenine,Adenosine, Adenosine diphosphate, Adenosine monophosphate, Adenosinetriphosphate, Cytidine, Cytidine diphosphate, Cytidine monophosphate,Cytidine triphosphate, Cytosine, Deoxyadenosine, Deoxyadenosinediphosphate, Deoxyadenosine monophosphate, Deoxyadenosine triphosphate,Deoxycytidine, Deoxycytidine diphosphate, Deoxycytidine monophosphate,Deoxycytidine triphosphate, Deoxyguanosine, Deoxyguanosine diphosphate,Deoxyguanosine monophosphate, Deoxyguanosine triphosphate, Guanine,Guanosine, Guanosine diphosphate, Guanosine monophosphate, Guanosinetriphosphate, Hypoxanthine, Inositol, Thymidine, Thymidine diphosphate,Thymidine monophosphate, Thymidine triphosphate, Thymine, Uracil,Uridine, Uridine diphosphate, Uridine monophosphate, Uridinetriphosphate.

A dispersed phase, e.g., an aqueous phase may comprise water and/or oneor more amino acids, amino acid derivatives, peptides, polypeptides,proteins/enzymes, co-factors, vitamins, salts, detergents, and/orbuffers.

Some preferred dispersed phase, e.g., aqueous phase formulations do notcomprise ionic detergents. Preferred dispersed phase fluids comprisenon-ionic detergents at concentrations lower than 5%, less than 0.5% ispreferred, 0.1% is even more preferred. Glycerol concentrations <20%,less than 10% preferred, <5% even more preferred. Total saltconcentration below 3 M, <1 M preferred. Total buffer concentrationhigher than 5 mM, higher than 10 mM preferred. An exemplary aqueousformulation for PCR may look like but is not limited to 20 mM Tris-HCl,2.5 mM MgCl2, 50 mM KCl, 0.06% IGEPAL® CA-630 (NP-40), 0.05% Tween® 20,25 mM NH4Cl, 200 uM each dNTP (dATP, dTTP/dUTP, dCTP, dGTP), (pH 8.9 @25° C.). This reaction would include a suitable polymerase listed in thelist herein.

Dispersed phase, e.g., aqueous phase formulations include any suitablereporter reagent as known by someone skilled in the art. See, e.g.,“Dyes,” above.

Partitions will demonstrate stability in a flow rate regime where theshear forces traveling through the system conduits are not larger thanthe interfacial tension stabilizing the partition surface. Acceptableshear forces may occur at partition velocities between 1 mm/sec and 75mm/sec, with preferred partition velocities between 4 mm/sec and 53mm/sec.

Fluoropolymers. In certain embodiments, one or more surfaces ofcomponents described herein, or, in some cases, entire components (e.g.,partitioners, etc.) may comprise a fluoropolymer. In these embodiments,any suitable fluoropolymer may be used. Exemplary fluoropolymers includepolytetrafluoromethylene (PTFE), chlorotrifluoroethylene (CTFE),polyvinylidene difluoride (PVDF), perfluoroalkoxy polymer (PFA),fluorinated ethylene-propylene (FEP), polychlorotrifluoroethylene(PCTFE), polyethylenetetrafluoroethylene (ETFE), ECTFE(polyethylenechlorotrifluoroethylene), FFPM/FFKM (PerfluorinatedElastomer [Perfluoroelastomer]), FPM/FKM (Fluorocarbon[Chlorotrifluoroethylenevinylidene fluoride]), FEPM (Fluoroelastomer[Tetrafluoroethylene-Propylene]), PFPE (Perfluoropolyether), PFFS(Perfluorosulfonic acid) or any combination thereof.

Thus, systems and methods as described herein may provide for accuratequantification or detection of biological material in a sample. In someinstances, systems and methods result in reduced contamination. Incertain further embodiments, reduction in contamination may be measuredby a frequency of amplification in droplets not comprising biologicalmaterial (e.g. DNA or RNA) and/or droplets comprising biologicalmaterial (e.g., DNA or RNA) from a sample other than the sample beinganalyzed. For example, reduction in contamination is measured indroplets generated from the dispersed phase comprising purging fluid orseparation fluid. In some instances, the frequency of amplification isat most or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 70%, or 75%. In some instances, the frequency of amplification isin a range of about 5% to about 75%, about 10% to about 70%, about 15%to about 65%, about 20% to about 60%, or about 25% to about 50%.Reduction in a contamination may be measured by a rate of falseamplification. In some instances, the rate of false amplification is atleast or about 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000,1:1250, 1:1500, 1:2000, 1:2500, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000,1:8000, 1:9000, 1:10000, 1:12000, 1:15000, 1:20000, 1:25000, 1:30000,1:40000, 1:50000, 1:60000, 1:70000, 1:80000, 1:90000, 1:100000,1:125000, 1:150000, 1:200000, 1:300000, 1:400000, 1:500000, 1:600000,1:700000, 1:800000, 1:900000, 1:1000000. In some instances, the rate offalse amplification is at most 1:300, 1:400, 1:500, 1:600, 1:700, 1:800,1:900, 1:1000, 1:1250, 1:1500, 1:2000, 1:2500, 1:3000, 1:4000, 1:5000,1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:12000, 1:15000, 1:20000,1:25000, 1:30000, 1:40000, 1:50000, 1:60000, 1:70000, 1:80000, 1:90000,1:100000, 1:125000, 1:150000, 1:200000, 1:300000, 1:400000, 1:500000,1:600000, 1:700000, 1:800000, 1:900000, 1:1000000.

Serial flow emulsion reactions such as quantification and detection ofnucleic acids or proteins are useful biological techniques. Traditionalmethods for quantification of samples, for example quantitativepolymerase chain reaction, require a number of amplifications to reach athreshold fluorescence intensity such that target nucleic acids in thesample may be detected. Digital reaction assays (e.g. digital PCR) arenot dependent on the number of amplification and/or reaction cycles todetermine the initial sample amount, eliminating the reliance onuncertain exponential data to quantify target nucleic acids andproviding absolute quantification. Further serial flow emulsionreactions allow for on-demand analysis and require less material.

Generally, digital assays require dividing a sample into partitions. Inthe serial flow emulsion reactions discussed herein, these partitionsare droplets. In a serial flow emulsion reaction system, samples to beassayed may be in the dispersed phase of the emulsion. These samples mayinjected serially (i.e. one-by-one) into the continuous phase of theemulsion and flowed through the steps necessary to partition, react, anddetect the results of the reaction in the samples. Such a system maylead to cross-contamination between samples. For example, individualvolumes of dispersed phase in the continuous phase can have a tendencyto coalesce. When partitioned droplets coalesce into larger droplets,the validity of the digital assay may be compromised. As anotherexample, axial dispersion of flowing partitioned droplets can causedroplets formed from a first sample to become interspersed with dropletsformed from a second sample. Such interspersion can cause the results ofthe digital assay for both samples to be invalid. The axial dispersioncan occur when individual sample injections are not temporally orspatially separated enough in the channel or tube containing theemulsion. The axial dispersion can also occur when dead zones exist inthe flow of emulsion, causing droplets formed from a first sample tohave a long enough residence time in the system that they becomeinterspersed with droplets formed from a second sample. As anotherexample, volumes of dispersed phase in the emulsion corresponding to afirst sample and a second sample can coalesce if they contact each otherprior to partitioning. Finally, cross-contamination may also result whenall or part of a dispersed phase volume strongly interacts with asurface of the channel or tube containing the emulsion, leading to longresidence times and possible incorporation of all or part of thedispersed phase volume by a subsequent sample or samples, leading to acompromised validity of assays on the subsequent sample or samples.

Provided herein are systems and methods for conducting reactions inserial flow of dispersed phase volumes of an emulsion, whereincross-contamination is minimized. In some instances, cross-contaminationoccurs as a result of incorporation of all or part of an individualdispersed phase volumes with one or more other dispersed phase volumes.Such cross-contamination can result in invalid assay results, undesiredreaction results or products, or incomplete separation or partitioningof elements in the emulsion (e.g. single cells). In some instances, thereactions of interest are assays to quantify a biological compoundcomprising individual dispersed phase volumes that represent separatebiological samples and/or reagents required to detect and/or quantifythe desired compound or compounds in the sample. In some instances, theassay is a digital assay. In some instances, the digital assay is adigital PCR assay to quantify one or more nucleic acids in the samples.Further systems and methods as provided herein may detect individualdroplets accurately. Accurate detection of individual droplets may occurby the reduction of cross-contamination in the system. Reduction ofcross-contamination may occur through prevention of wetting of systemsurfaces by volumes of dispersed phase, droplets becoming stuck onsurfaces of the system or slowed by dead-zones in system flow,decontamination of surfaces that have been exposed to biological orchemical samples, separation of individual droplets with a fluid,separation of groups of droplets in a fluid originally produced fromdistinct reaction mixtures, samples, or assay mixtures, or detection ofan individual droplets in serial flow. Further described herein aresystems and methods that allow for reaction multiplexing using spatialand temporal techniques, where multiplexing refers to conductingmultiple reactions or assays on the same volume of reaction mixture,sample, or assay mixture. Systems and methods as described herein maynot require plugs for achieving desired reaction results, accurateassays, or accurate detection of individual droplets. Systems andmethods described herein may allow reactions in serially flowingdispersed phase volumes of an emulsion to be conducted without therequirement that those volumes have substantially the same cross sectionas the tube or channel containing the emulsion.

Described herein are systems and methods for serial flow emulsionreactions. In some instances, the systems comprise a sampling device, aninjector, a reactor, and a detector. In some instances, the samplingdevice pulls in either reaction mixtures or samples to be assayed. Insome instances, the reaction mixtures or samples to be assayed are in adispersed phase. In some instances, the dispersed phase comprisingreaction mixtures or samples to be assayed are referred to as a firstdispersed phase. The samples may be pulled in through a tube or achannel comprising a material with low affinity or surface energy forthe first dispersed phase and a higher affinity or surface energy forthe continuous phase of the emulsion. The first dispersed phase maycomprise an aqueous phase solution of water, PCR mastermix (buffers,salts, dNTPs), primers, probes, and nucleic acid molecules (e.g. DNA orRNA). In some instances, the first dispersed phase comprises a solutionof reactants, markers, and protein. In some instances, the firstdispersed phase comprises a chemical species or a nanoparticulate of achemical species for chemical synthesis.

Described herein are methods and systems for serial flow emulsionreactions, wherein the serial flow emulsion reactions may be performedon a sample comprising at least one nucleic acid. In some instances, thesample comprises multiple nucleic acids. Exemplary nucleic acidsinclude, but are not limited to, coding or non-coding regions of a geneor gene fragment, intergenic DNA, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA,ribozymes, complementary DNA (cDNA), DNA molecules producedsynthetically or by amplification, genomic DNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, or isolated RNA of any sequence. In some instances,the sample comprises DNA. In some instances, the sample comprises RNA.

Samples as described herein may further comprise one or more reagentsfor performing a reaction. In some instances, the reaction is a nucleicacid amplification reaction. For example, the nucleic acid amplificationreaction is polymerase chain reaction (PCR). Non-limiting amplificationreactions include, but are not limited to, PCR, quantitative polymerasechain reaction (qPCR), self-sustained sequence replication,transcriptional amplification system, Q-Beta Replicase, or rollingcircle replication. In some instances, PCR comprises digital PCR indroplets. Exemplary reagents for a nucleic acid amplification reactioninclude, but are not limited to, enzymes (e.g. polymerase,transcriptase), buffers, dNTPs, primers, or probes. In some instances,the sample comprises an intercalating dye, probes, or molecular beacons.

In some instances, the first dispersed phase comprises oils. In someinstances, the first dispersed phase is dispersed phase in anoil-in-water emulsion. In some instances, the dispersed phases combinewith a continuous or semi-continuous flow of a continuous phase to forma flowing emulsion. An “emulsion” may be referred to as a two-phasemixture of a dispersed phase in a continuous phase. In some instances,the continuous phase is hydrophobic. In some instances, the continuousphase comprises a hydrophobic oil. In some instances, the hydrophobicoil is a fluorinated oil. In some instances, the continuous phase ishydrophilic. In some instances, surfaces of the injector or one or morechannels are fluorinated. In some instances, the surfaces of theinjector or one or more channels are hydrophilic.

II. Intake System

Systems and methods provided herein can include an intake system and aprocess system, where the intake system and process system areconfigured so that they are not in continuous fluid connection, but adispersed phase, e.g., a sample, taken up by the intake system from asuitable dispersed phase container, e.g., sample container, or series ofdispersed phase containers, e.g., sample containers, can be moved fromthe intake system to the process system. This can be accomplished in anysuitable manner. In certain embodiments, an injector serves as aninterface between the intake system, also referred to herein as asampler, autosampler, or similar wording, and the process system, wherethe injector can cycle between a configuration that is in fluidcommunication with the intake system and a configuration that is fluidcommunication with the process system. The injector can be configured tohave additional configurations, e.g., configurations that allow cleaningof one or more parts of the intake system and injector. In certainembodiments, the injector comprises common conduit (also referred toherein as an injection chamber, or injection loop) where the commonconduit can be in fluid communication with the intake system or in fluidcommunication with the process system, but cannot simultaneously be influid communication with both. Thus, the intake system and/or theinjector can be treated between injections of dispersed phase, e.g.,between samples, in order to reduce or eliminate and/or rendernon-reactive, traces of dispersed phase, e.g., sample, between rounds ofintake of dispersed phase, e.g., sample. It will be appreciated that“intake system” can include the injector when the system is configuredto be fluidly connected to the injector, as will be clear from contextin the following description.

Thus, systems and methods as described herein for serial flow emulsionreactions comprise a sampling device, or sampler, also referred to as anintake system herein. In some instances, the sampling device is used tointroduce one or more samples into systems as described herein.

Described herein are systems and methods for serial flow emulsionreactions comprise use of a sampling device, wherein the sampling devicecomprises a staging container (e.g., sample container, or plurality ofsample containers). The staging container may be a microwell plate, astrip of PCR tubes, or a single PCR tube. In some instances, themicrowell plate has at least or about 24 wells, 48 wells, 96 wells, or384 wells. In some instances, the microwell plate has at least or about12 wells, 24 wells, 36 wells, 48 wells, 60 wells, 72 wells, 96 wells,108 wells, 120 wells, 240 wells, 384 wells, 768 wells, 1536 wells, ormore than 1536 wells. In some instances, the fluid injector selects aspecific sample from the staging container by physically moving anintake portion of the fluid injector to a geometric position of thestaging container that contains the specific sample. In some instances,the fluid injector selects a specific sample from the staging containerby changing the state of a multiport valve in fluid communication withat least two geometric zones of the staging container containing adistinct sample within each geometric zone.

In some instances, each well of the staging container comprises at leastor about 100 nL, 200 nL, 300 nL, 400 nL, 500 nL, 600 nL, 700 nL, 800 nL,900 nL, 1000 nL, 2000 nL, 3000 nL, 4000 nL, 5000 nL, 6000 nL, 7000 nL,8000 nL, 9000 nL, 10000 nL, 20000 nL, 30000 nL, 40000 nL, 50000 nL,60000 nL, or more than 60000 nL. In some instances, each well of thestaging container comprise at least 10 uL, 20 uL, 50 uL, 100 uL, 200 uL,or more than 200 uL.

Cleaning routines. The intake system can include an intake line, alsoreferred to herein as an intake channel, intake conduit, aspirationconduit, or similar wording for moving sample from a sample container toan injector, and the portions of the injector that are exposed tosample, for example, a common conduit. The intake line can undergo acleaning cycle between samples.

The purpose of a cleaning cycle on the intake system is to: 1) Purge anycontaminant, such as detectable (before or after amplification)material, from the intake section of the system; 2) Render any materialnot purged from the system undetectable; and/or 3) Inject a spacer fluidin between each of the samples so that the partitions from one sample donot intermingle with the partitions from the other sample. In practice,this can include of a variety of sequences. These can include one ormore of purge, denature, and/or space. Possible sequences include: purgeonly; denature and purge; purge and space; denature, purge, and space.

In certain embodiments, an aliquot of a first fluid, such as dispersedphase, e.g., a sample, is transported from a container via an intakeline to an injection chamber (common conduit) in an injector (generallyconsidered part of the intake line when the system is configured toallow a fluid connection between the injector and the intake system) isinjected from the injector into the process side. A certain portion ofthe first fluid is moved from the intake line to the process side inthis step, e.g., at least 60, 70, 80, 90, 95, 99, or 99.5% of the firstfluid. In certain embodiments, at least a second fluid is moved throughpart of all of the intake line, such as a purge fluid, denaturing fluid,spacer fluid or any other suitable fluid as described herein. After thisstep, at least 60, 70, 80, 90, 95, 99, or 99.5% of any remaining firstfluid in the intake line is removed. In certain embodiments, a thirdfluid is moved through part of all of the intake line, such as a purgefluid, denaturing fluid, spacer fluid, or any other suitable fluid asdescribed herein. After this step, at least 60, 70, 80, 90, 95, 99, or99.5% of any remaining first fluid in the intake line is removed. Anysuitable number of such steps can be performed, where after each step atleast 60, 70, 80, 90, 95, 99, or 99.5% of any remaining first fluid inthe intake line is removed.

Purging can be done by effusion or by a mixture of effusion andpreferential chemical compatibility. In certain embodiments, suction iscreated at the intake point to pull material into an intake line. Theintake line can be any suitable intake line, for example, a tube or aneedle. The intake line, e.g., tube can be made of fluoropolymer (e.g.PTFE, CTFE), polymer (e.g. nylon, polyethylene, etc.), metal (e.g.stainless steel, aluminum, etc.), or any other suitable material. Incertain embodiments, surfaces of the intake line that come in contactwith sample have greater affinity for one or more purge, denaturing,and/or spacer fluids (or other fluids, e.g., dead-volume fluids asdescribed below) than for sample, e.g., sample in an aqueous phase.

In the course of sampling, the intake line can retain contaminatingmaterial, including detectable or amplifiable material from the sample(e.g. DNA, RNA, cDNA, proteins, etc.), or any other material that couldaffect one or more operations on the process side in such a way as toalter or potentially alter a process operation in such a way as tomaterially affect results from the process, e.g., results from one ormore other samples. This material may be suspended or solvated inaqueous phase (e.g. as part of a sample or aqueous residue from asample), and/or it may be physically or chemically bound to the surfaceof the tube. If this material is incorporated into a subsequent sampleinjection into the process portion of the system, it could provide falseor biased results. For PCR-based reactions, even a single molecule ofamplifiable material can be detected, so it is important to eitherremove all or substantially all such material between samples or tocompletely denature any material that is remaining. By denature, it ismeant that the material is incapable of or substantially unlikely to beinvolved in processes on the process side, e.g., reactions; in the caseof PCR, this includes incapable or substantially unlikely either beamplified or detected or both.

The purpose of purging is to force material that does not containcontaminating material, such as detectable or potentially detectablematerial, through the intake system, displacing any contaminatingmaterial, such as detectable or potentially detectable material, fromthe intake system so that it does not get injected into the processportion of the system. In one basic element, the purging fluid compriseswater. However, the purging fluid can also comprise other materials,e.g. fluorinated or perfluorinated oils, silicone oils, organic oils,mineral oils, acids/bases, detergents, combinations thereof, or anyother suitable material.

To improve the efficacy of purging, the purging fluid can comprise afluid that has a higher affinity for the material of construction of thesurface of the intake line, than water, water soluble-compounds, ordetectable/potentially-detectable material. In these cases, the higheraffinity of the purging fluid acts to displace any sample fluid orresidual contaminating material, e.g., detectable/potentially detectablematerial, from the intake line so that it will not be incorporated intoa subsequent sample. In certain embodiments, the surface of the intakeline comprises a fluoropolymer and the purging fluid comprises afluorinated oil. The purging fluid can also comprise a hydrophobicmaterial if the surface of the intake line is comprised of hydrophobicmaterial. Alternatively, when the sample comprises a hydrophobicmaterial, the purging fluid could comprise a hydrophilic material andthe intake line could comprise a hydrophilic material. A purging stepmay comprise flowing more than one purging fluid through the intakeline, such as at least two, three, or four purging fluids.

The purpose of denaturing is to chemically or physically alter anycontaminating material, such as detectable or potentially detectable(e.g. detectable after an amplification reaction) material, so that itis no longer contaminating, e.g., no longer detectable or potentiallydetectable. By following a sample intake step with a denaturing fluid,residual contaminating material, e.g., detectable or potentiallydetectable material, may not cross-contaminate future samples.Denaturing agents can include any suitable agent or combination ofagents, depending on the nature of the sample, e.g., acids/bases,peroxides, bleach, DNA modifying enzymes, intercalating agents, and thelike. In certain embodiments where the sample comprises nucleic acids,e.g., nucleic acids to be amplified by PCR, the denaturing fluid may beany fluid that may prevent the nucleic acid molecule from beingamplified. In some instances, the decontamination fluid comprises anaqueous solution comprising, e.g., azides, hypochlorite, e.g., sodiumhypochlorite, mineral acids such as phosphoric acid, strongly alkalinesolutions, e.g., sodium hydroxide, peroxides, RNAse, or DNAse, or acombination thereof. In some instances, the denaturing fluid comprisesat least or about 0.5%, 1.0%, 1.5%, 2.0%, 4.0%, 6.0%, 8.0%, 10%, 12%,14%, 16%, 18%, 20%, 24%, 28%, 32%, 34%, 36%, 40%, 44%, 50%, 60%, 70%,80%, 90%, or more than 90% of azides, hypochlorite, e.g., sodiumhypochlorite, mineral acids such as phosphoric acid, strongly alkalinesolutions, e.g., sodium hydroxide, peroxides, RNAse, or DNAse, or acombination thereof.

Thus, in the case of nucleic acids, the denaturing (decontamination)fluid for use in the sampling device as described herein may comprise atleast one chemical component that changes the chemical and/or physicalcomposition of nucleic acids such that they can no longer undergo areaction. In some instances, the reaction is polymerase chain reaction(PCR). In some instances, the wetted surfaces of the fluid injectorand/or injection device are exposed to the at least one chemicalcomponent. In some instances, any cross-contamination from previousinjected nucleic acid molecules is reduced or eliminated. In someinstances, the denaturing (decontamination) fluid comprises water. Insome instances, the decontamination fluid comprises at least onechemical component from the group consisting of sodium hypochlorite,phosphoric acid, sodium hydroxide, RNAse, or DNAse. In some instances, aconcentration of the at least one chemical component is at least orabout 0.5%, 1.0%, 1.5%, 2.0%, 4.0%, 6.0%, 8.0%, 10%, 12%, 14%, 16%, 18%,20%, 24%, 28%, 32%, 34%, 36%, 40%, 44%, 50%, 60%, 70%, 80%, 90%, or morethan 90%.

The purpose of a spacing fluid, also referred to as spacer fluid,separation fluid, and similar terms herein, is to provide a breakbetween samples so that partitions from one sample flowing through thesystem are not interspersed with partitions from a second sample. Incertain embodiments, the spacing fluid comprises water and does notcomprise detectable or potentially detectable material. In certainembodiments, the spacing fluid may or may not comprise a surfactant tostabilize partitions comprising water flowing in a hydrophobiccontinuous phase; suitable surfactants can be, e.g., as describedherein. In certain embodiments, the spacing fluid comprises an oil, suchas silicone oil, organic oil, mineral oil, or a combination thereof, forexample, mineral oil. In certain embodiments the spacing fluid comprisesa material that is substantially immiscible with the sample and with thecontinuous phase; in certain embodiments the spacing fluid comprises amaterial that has a greater affinity for the surface of one or moreconduits in the intake system or process system than do one or more ofthe samples. The physical size of the spacing fluid volume preventspartitions from a first sample injected at a first time into the processportion of the system from becoming interspersed with partitions from asecond sample injected at a second time subsequent to the first time;even though it is expected some axial dispersion of partitions willoccur, the scale of the dispersion will not be as large as the linearsize of the volume of spacing fluid injected, so no partitions willintersperse. Additionally, since the spacing plugs are exceptionallylarge so that they consume the entire or substantially the entirecross-sectional diameter of flow conduit on the process side, partitionshave a limited probability of transiting. While simply using an aqueousspacing fluid can work, problems may arise if partitions become trappedin regions of flow moving slower than the bulk flow (e.g. dead zones,eddies, and the like). Additionally, in regions of flow where there is achange in the vertical position of the partitions, relative differencesin partition velocity as a function of size due to buoyancy can causeaxial dispersion to be more severe. For these reasons, using a spacingfluid comprising a material immiscible with water is advantageous. Ifthat material has a higher affinity for the material comprising the flowconduit, it can displace and force partitions in slow moving zones tomove through the system. Similarly, if the material has a high viscositythe material will tend to exhibit a low deformation rate acting as astrong physical front that may displace materials from the conduit wallsand prevent partition transit.

The order in which sample materials are added can be important toprevent cross-contamination in this system. The intake and processsections of the system can be separated by an injector. The injector isan element of the system that allows independent intake and thenprocessing of fixed samples from that intake. In certain systems andmethods provided herein, a “common conduit” approach (describedelsewhere) is used—this allows separation of the intake and the processsections of the system by physically moving an inline section (the“common conduit”) of tubing from the intake section to be inline withthe process section. In certain embodiments, the surface of the commonconduit comprises a material that has a higher affinity for thecontinuous phase of the emulsion to be created in the process side ofthe system. Thus, when said continuous phase is added to the commonconduit, it will preferentially displace any dispersed phase from thesystem. This can be important to cleaning the system to preventcross-contamination. In certain embodiments, the common conduit surfacecomprises a hydrophobic material, the continuous phase comprises ahydrophobic component, and the dispersed phase of the emulsion comprisesa hydrophilic component.

Purge only: A sample is pulled into the intake section of the system andat least partially fills the injector's common conduit. The injectormoves the common conduit to be in fluid communication with the processsection of the system and the sample exits the common conduit towards,e.g. the partitioner. When the elements of the injector that contactedthe sample are realigned with the intake section of the system, analiquot of purge fluid is pulled into the system. The aliquot is largeenough so as to completely displace any residual sample fluid that maycontain contaminating material from the common conduit and intakesystem, such as detectable or potentially detectable material. Two ormore aliquots may be sampled of two or more different purge fluids. Incertain embodiments, the purge fluid comprises a material with a greateraffinity for the conduit surface than does the material to be sampled,so as to displace any residual sample material from the common conduitand the intake system. In certain embodiments, a first purge fluidcomprising water may be added to the system, displacing residual sampleto injector waste. A second purge fluid comprising a hydrophobicmaterial with higher affinity for the conduit surface than water maythen be sampled, displacing any residual water and preparing the systemfor a new sample. In certain embodiments the second purge fluidcomprises an oil, such as a fluorinated oil.

Denature and purge: This is the same as purge only, except an additionalstep or series of steps is added where denaturing fluid is pulled intothe system ahead of the purge fluid or fluids. For example, a sample maybe pulled into the common conduit of the injector, which is thenpositioned to be in fluid communication with the process system and thesample displaced by at least one continuous phase. The common conduit isthen positioned to be in fluid communication with the intake system, andan aqueous solution comprising bleach is flowed through the commonconduit so as to render nucleic acid in the intake system unable to beamplified, followed by a fluorinated oil to displace the aqueoussolution comprising bleach from the common conduit and intake system.

Purge and space: This is the same as purge only, except in betweensequences of sample injections, a spacing fluid is injected into theprocess system. A typical sequence can be, e.g., starting with aninjector filled with purge fluid: Sample intake into injector, injectinto process system, purge fluid(s) intake into injector, reject towaste, spacing fluid intake into injector, inject into process system,purge fluid(s) intake into injector, reject to waste.

Denature, purge, and space: This is the same as “Purge and space”,except a denaturing fluid is injected ahead of the purging fluid in atleast one part of the sequence. Such a sequence can be, e.g., startingwith an injector filled with purge fluid: Sample intake into injector,inject into process system, denaturing fluid intake into injector,reject to waste, purge fluid(s) intake into injector, reject to waste,spacing fluid intake into injector, inject into process system, purgefluid(s) intake into injector, reject to waste. An alternate sequencecan be, e.g.: Sample intake into injector, inject into process system,denaturing fluid intake into injector, reject to waste, purge fluid(s)intake into injector, reject to waste, spacing fluid intake intoinjector, inject into process system, denaturing fluid into injector,reject to waste, purge fluid(s) intake into injector, reject to waste.

If the inlet region of the intake line comprises a filtering element toreject or partially reject particulate matter above a certain size(generally, above some fraction of a critical dimension of the smallestelement of the microfluidic system), then a “blowback” step may be addedto any of the steps in the sampling sequence. In such a step, fluid flowmay be reversed so as to, e.g., dislodge particulate material from thesurface of the inlet region of the intake channel. This blowback stepcan be added after intake of either the denaturing or the purge fluid,but it can be added to any step in the process. The fluid rejected canbe rejected into a waste receptacle. Preferably, the fluid rejected willbe of low value.

In certain embodiments, the denature, purge and space fluids areinjected using the sampling nozzle and are passed to either into theprocess section of the system (in the case of the spacing fluid) or towaste (in the case of purge and denature fluids). In certain embodimentthe denature, purge and/or space fluids are pulled from fluid reservoirs(such as bottles, bags, and the like) and are passed in the reversedirection through the injector and sampling nozzle disposing into thenow empty sample container, such as sample well, or into a separatecontainer or well designated for waste.

It may be necessary to rinse the outside of the sample nozzle to removeany adsorbed or otherwise adhering liquids. This is especiallyproblematic when working with viscous agents. The rinse may be done inany suitable manner, such as by wiping, dipping in a lower viscositysolution, or by rinsing the tip with continuous phase using an externalstream of continuous phase.

Thus, in certain embodiments, sampling device may comprise a stagingcontainer, a pump, a decontamination fluid reservoir, a purge reservoir,and a sampler intake. The staging container may hold at least one sampleto be analyzed on the instrument. The pump may provide the motive forcerequired to move the sample fluid from the staging container to theinjector. The decontamination fluid reservoir may comprise fluid fordecontaminating the system between injections of samples from one ormore staging containers. The purge reservoir may comprise a dispersedphase for separating each sample in the reaction flow pathway. The fluidinjector may transfer sample, decontamination fluid, or the dispersedphase from the staging container to the injector. A controller maycontrol the sequence and timing of events during sample loading andinjection.

Thus, provided herein are systems and methods for serial flow emulsionreactions, wherein the sampling device introduces one or more samplesand one or more dispersed phases in sequence. At the start of thesequence, an internal volume of the fluid injector (also referred to asan intake system, sampler, and other similar terms herein) may comprisedecontamination (denaturing) fluid, second dispersed phase, aqueoussolution not comprising nucleic acid, denatured nucleic acid, orcombinations thereof. The fluid injector may select a sample from thestaging device. The controller may activate the pump and may control thepump rate and activated time such that a first controlled volume ofsample is loaded by the fluid injector into the injection device (alsoreferred to herein as injector). Once complete, the controller may stopthe pump and may direct the fluid injector to inject the decontaminationfluid from the decontamination fluid reservoir into the injectiondevice. The controller may start the pump and may control the pump rateand activated time such that a second controlled volume ofdecontamination fluid may loaded by the fluid injector into theinjection device. Once complete, the controller may stop the pump andmay direct the injector to inject the second dispersed phase from thepurge fluid reservoir into the injector. The controller may start thepump and may control the pump rate and activated time so that a secondcontrolled volume of separation and/or purge fluid may be loaded by thefluid injector into the injection device. Once complete, the controllermay stop the pump and the system may ready to load another sample fromthe staging container.

In some instances, the sequence is varied. At the start of the sequence,the internal volume of the fluid injector may comprise decontaminationfluid, second dispersed phase, aqueous solution not comprising nucleicacid, denatured nucleic acid, or combinations thereof. The fluidinjector may select a sample from the staging device. The controller mayactivate the pump and may control the pump rate and activated time sothat a first controlled volume of sample is loaded by the fluid injectorinto the injection device. Once complete, the controller may stop thepump and may direct the fluid injector to inject the second dispersedphase from the purge fluid reservoir into the injection device. Thecontroller may start the pump and may control the pump rate andactivated time so that a second controlled volume of decontaminationfluid may be loaded by the fluid injector into the injection device. Insome instances, decontamination fluid and first dispersed phase areprevented from intermingling. Once complete, the controller may stop thepump and may direct the fluid injector to inject the second dispersedphase from the purge fluid reservoir into the injection device. Thecontroller may start the pump and may control the pump rate andactivated time so that a second controlled volume of decontaminationfluid is loaded by the fluid injector into the injection device. Oncecomplete, the controller may stop the pump and the system may be readyto load another sample from the staging container. In some instances,the controller stops the pump and directs the fluid injector to injectthe second dispersed phase from the purge fluid reservoir into theinjection device. In some instances, the controller again the pump andcontrols the pump rate and activated time so that a second controlledvolume of decontamination fluid is loaded by the fluid injector into theinjection device before loading another sample from the stagingcontainer.

Dead volume fluid. Because the intake channel is of finite size andcomprises a pathway to the injector, the total volume of the system willof necessity be larger than the volume injected in the injector. If thevolume of the intake channel significantly exceeds the volume of thematerial to be sampled or a desired volume of any of the cleaningreagents, it may be desirable to add a sampling step or steps of a “deadvolume fluid.” This fluid occupies the dead volume of the system outside(or even partially comprising) the volume of the common conduit of theinjector. In one embodiment, the dead volume fluid is air. Using air asthe dead volume has the advantage of being free and of unlimited supply,and the interface of air with sample is easily detectable (see below);on the other hand, it is compressible, so is more difficult to meterinto the system to ensure accurate loading of the injector. However,since the air-fluid interface is easily detectable using common methods,including but not limited to ultrasonic or optical methods, detectorsmay be used to ensure the placement of the sample into the commonconduit. These detectors may provide additional functionality such assample loading verification in the case of limited/no sample injectionas well as a means for quantifying the volume of sample loaded into thecommon conduit. This may be derived since the flow rate and tubedimensions are fixed and therefore injected volume is a function oftransit time of the sample through the air detector.

The dead volume fluid can comprise a material in the continuous phase ofthe emulsion to be created in the process portion of the system. Forexample, the dead volume fluid can be a fluorinated oil, a silicone oil,a hydrocarbon oil, an organic oil, or an aqueous fluid; in certainembodiments the dead volume fluid comprises a fluorinated oil. More thanone dead volume fluid may be used in the system. See, e.g., “parfait”systems and methods, described below.

Dead volume fluid may be added to the intake line in any suitablemanner. In certain embodiments, dead volume fluid is contained in anonboard reservoir that is accessible by the sampling head of the intakechannel. After pulling sample from a sample container into the intakechannel, the sampling head is repositioned so that the intake channelcan pull the at least one dead volume fluid into the intake channel fromthe common reservoir. While this approach is simple, it riskscontaminating the dead volume fluid with contaminating material, such asdetectable or potentially detectable material, from previous samplecontainers sampled and additionally requires regular refilling. Incertain embodiments, this can be prevented by having a plurality of deadvolume fluid reservoirs; in the limiting case, there is one dead volumefluid reservoir for each sample container, which eliminates thepotential for cross-contamination. In this embodiment, dead-volume fluidcould be provided in a pre-filled consumable (e.g. a sealed microtiterplate) or a user-filled consumable and placed in a position accessibleto the sampling head. Alternatively or additionally, there can be a setof fillable and cleanable reservoirs on the system that are filled froma central dead fluid reservoir. These reservoirs can be filled using agravity feed, a pump system, or any suitable means to achieve fluidflow. In some cases, all of the dead volume fluid dispensed is pulledinto the intake channel. In other cases, only a portion of the deadvolume fluid is pulled into the intake channel, and the balance isremoved through a drain in the dead volume fluid reservoir. A variety ofconfigurations can be envisioned for valves, pumps, and drains toachieve this approach.

In certain embodiments, dead volume fluid may be dispensed intoreservoirs through a channel attached to the sampling head. This may bethe same channel as the intake channel or a separate channel;embodiments of these approaches are described further below. In somecases, a reservoir or reservoirs are filled with dead volume fluidbetween each intake of sample. For example, there can be a dedicateddead volume container; after pulling sample from the at least one samplecontainer (e.g. a well of a microtiter plate), the dispensing channel ofthe sampling head can be positioned into the dead volume fluid reservoirand dead volume fluid can be pulled into the reservoir. Any suitablemethod of generating a driving force may be used, e.g., the use ofperistaltic or syringe pumps to create a negative pressure environment.In another example, the well of the microtiter plate that contained thesample can be used as the dead volume reservoir. After pulling sampleinto the intake portion of the system, a second channel can bepositioned over the well and dead volume fluid may be dispensed throughthat channel. The first channel may then be positioned over the well anddead volume fluid pulled in immediately behind the sample. In anotherexample, the dead volume fluid is dispensed into wells in a microtiterplate that may be used a finite number of times before disposal, but aredistinct from the wells that held the original sample.

In another embodiment, the dead volume fluid may be supplied through abranch channel to the intake channel but ahead of the injector. In suchan embodiment, a three-way valve or similar device can select betweenthe sample intake channel and the dead volume fluid channel as inlets tothe injector. After intake of sample (potentially followed by air) andpositioning of that sample in or substantially in the injector, thevalve position can be changed so that intake of further fluid comes fromthe dead volume fluid reservoir.

In certain embodiments, the reservoir holding the dead volume fluid isthe sample container itself (e.g. a well in a microtiter plate), and thedead volume fluid is separated from the immiscible sample fluid bygravity, electromagnetic force, or any other suitable force. In certainembodiments, the dead volume fluid and the sample fluid have differentmass densities and gravity allows the dead volume fluid to settle outfrom the sample fluid. In certain embodiments, the dead volume fluid hasa lower mass density than the sample fluid and floats on top of the deadvolume fluid. The intake channel is inserted into a position of thesample fluid and intake started. As fluid is drawn into the intakechannel, the level of the top of the fluid in the sample container fallsuntil a transition is made to intake of the dead volume fluid due tocomplete injection of the sample fluid originally at a vertical positionat or above the vertical position of the inlet of the intake channel. Inthis way, sample fluid and dead volume fluid can be added withoutrepositioning the intake channel outlet or introducing air. In certainembodiments, the sample fluid may have either a lower gravimetricdensity or a higher gravimetric density as the dead volume fluid (butnot the same), so that one of the fluids rests on top of the other fluidwhen settled under a gravitational force. The intake channel inlet ispositioned so that it is located within or substantially within thesample fluid; a first volume of sample fluid is pulled into the intakechannel; the intake channel inlet is next positioned so that it islocated in or substantially in the dead volume fluid; a second volume ofdead volume fluid is pulled into the intake channel. In this way, deadvolume fluid can be injected immediately after sample fluid.

In the above set of embodiments, dead volume fluid can be added to thesample container containing sample fluid in any suitable manner. Forexample, the system user can prepare the fluids in the sample containerexternally to the system, e.g., the user can pipette sample fluid anddead volume fluid together into a well of a microtiter plate by hand.Alternatively or additionally, a liquid handling robot can perform thepipetting of either or both fluids. The fluids can be added in eitherorder. In another example, the dead volume fluid can be dispensed by thesystem into the sample container, either through a channel distinct fromthe intake channel or through the intake channel itself. In the formerinstance, dead volume fluid can be driven from a dead volume fluidreservoir by an appropriate driving force through the supply channel andinto the sample container. In the latter instance, dead volume fluid canbe driven in the reverse direction of flow as sample intake through theintake channel by utilizing a valving arrangement either ahead of orbehind the injector.

In these embodiments, separation of the fluid by gravity is importantfor proper functioning of the system. While the immiscibility of thedead volume fluid and the sample fluid will tend to make distinct phaseseparation of the system a stable equilibrium point, metastable statescould exist where portions of one phase are suspended in the otherphase. Agitating the two-phase system may be beneficial to phaseseparation. The velocity of the second fluid added may create enoughturbulence to agitate the fluid and be sufficient to drive separation.In certain embodiments, mechanical force may be used to agitate thefluid and drive separation; this can be any suitable force, e.g.,vibration, centrifugation, ultrasonic waves, or other methods thatagitate the fluids in the internal mixture. In certain embodiments, theinlet of the intake channel may be moved vertically, horizontally, or acombination of these motions so as to mechanically stir or agitate thefluids inside the sample container and encourage phase separation.

More than one dead volume fluid may be used in the system. In someembodiments, a first dead volume fluid is a spacer fluid and a seconddead volume fluid is a continuous phase or air. The common conduit ofthe injector is partially filled with sample then partially filled withthe first dead volume fluid; the balance of the intake system ispartially or fully filled with the second dead volume fluid. Taking thisapproach allows simultaneous injection of a sample fluid and asubsequent spacer fluid into the process system. In other embodiments,the system uses at least two dead volume fluids, where one of the deadvolume fluids comprises air. Air and at least one additional dead volumefluid may be added to the intake system in any order and any number ofaliquots. In an embodiment, air is added following intake of a samplefluid, followed by a second dead volume fluid, followed again by air. Incertain embodiments, the second dead volume fluid comprises an oil, afluorinated oil, a hydrocarbon oil, an organic oil, or a silicone oil.

Thus, systems and methods provided herein may include an exemplarysystem such as illustrated in FIG. 84. The system 101 comprises a firstdispersed phase 103 comprising samples, for example comprising a nucleicacid molecule and/or reagents for performing a nucleic acidamplification reaction, and a second dispersed phase 105 comprising afluid to prevent or eliminate cross-contamination. It will beappreciated that, in some instances, “dispersed phase,” as that term isused herein, may include materials that are moved into a sampler and/orinjector, such as sample, purge fluid, decontamination (e.g.,denaturing) fluid, and the like; in some instances such substances donot form an emulsion in a continuous phase. Usage of the term is clearfrom context. In some instances, the first dispersed phase comprises areaction mixture or reaction fluid. In some instances, the fluid is adecontamination fluid (also referred to as a denaturing fluid herein), apurge fluid, separation fluid (also referred to as spacer fluid herein),or a combination thereof. The decontamination fluid may be a fluid thatprevents a reaction from occurring. The purge fluid may be any fluidthat displaces any residual volume of the first dispersed system. Insome instances, the purge fluid does not affect, interfere, or confoundany measurement detected by the detector. The separation fluid (alsoreferred to as “spacer fluid” herein) may be a fluid that acts as aphysical buffer between two subsequent injections of a first dispersedphase. In some instances, the fluid is injected alternately with thefirst dispersed phase. The first dispersed phase 103 comprising samplesand a second dispersed phase 105 pass to a sampling device (“sampler”)107 and then an injector 109. The sampling device may function tochoose, transport, and/or potentially meter volumes of the variousdispersed phases. The injector may insert or introduce a volume ofdispersed phase into the continuous phase to form a volume of dispersedphase in the flowing emulsion. A continuous phase from a continuousphase reservoir 111 may be added to the injector 109. From the injector109, the first dispersed phase 103 comprising samples and a seconddispersed phase 105 pass to a reactor 113 and a detector 115. Thereactor may cause a reaction on the various volumes of first dispersedphase, for example, through heat, light, or acoustic energy.

Samples as described herein may be in volumes of one or more dispersedphases. In some instances, a dispersed phase is aqueous. In someinstances, a dispersed phase comprises more than about 51% (by mass orby molar concentration) of water. In some instances, a dispersed phasecomprises at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or more than 95% water. In some instances, the reagent or targetmolecule is encapsulated within an aqueous fluid. In some instances, theencapsulated reagent or target molecule is mixed with an immisciblefluid to form an emulsion. In some instances, the emulsion is a singleemulsion, a double emulsion, or a rod-like emulsion. In some instances,the immiscible fluid is oil. Exemplary oils are fluorinated oils,silicone oils, hydrocarbon oils, or mineral oils. In some cases, theimmiscible fluid comprises oil and one or more surfactants; surfactantcan be any suitable surfactant in any suitable concentration, forexample, as described herein. In some instances, a ratio of a volume ofthe droplet (partition) to the immiscible fluid (continuous phase) is atleast or about 1:20, 1:15, 1:10, 1:5, or 1:1. In some instances, theratio of the volume of the droplet to the immiscible fluid is about1:10. In some instances, the ratio of the volume of the droplet to theimmiscible fluid is about 1:1, for example, 0.5:1 to 1.5:1. Sometimes areagent or target molecule (e.g. DNA or RNA) is encapsulated in dropletsof dispersed phases.

Systems and methods as described herein may comprise a plurality ofdispersed phases. FIG. 85 shows a system 201 comprising three dispersedphases. A first dispersed phase 203 comprises a sample, for examplecomprising a nucleic acid molecule and/or reagents for performing anucleic acid amplification reaction. The first dispersed phase 203 issampled and injected and a portion may be rejected to waste. A seconddispersed phase 205 comprises a decontamination fluid. The seconddispersed phase 205 is sampled but not injected. The decontaminationfluid may be any fluid that may prevent the nucleic acid molecule frombeing amplified when the sample comprises nucleic acid. In someinstances, the decontamination fluid comprises sodium hypochlorite,phosphoric acid, sodium hydroxide, RNAse, or DNAse, or a combinationthereof. The third dispersed phase 207 comprises a separation (spacer)fluid. The third dispersed phase may provide a buffer between the firstdispersed phase and the second dispersed phase. The third dispersedphase may be sampled and injected and a portion may be rejected towaste. In some instances, the third dispersed phase comprises water. Insome instances, the third dispersed phase comprises an immiscible fluid.The first dispersed phase 203 comprising samples, a second dispersedphase 205, and a third dispersed phase 207 pass to a sampling device(“sampler”) 209 and then an injector 211. A continuous phase from acontinuous phase reservoir 213 may be added to the injector 211. Fromthe injector 211, the first dispersed phase 203 comprising samples, asecond dispersed phase 205, and a third dispersed phase 207 pass to areactor 215 and a detector 217.

FIG. 86 shows a system 301 comprising four dispersed phases. A firstdispersed phase 303 comprises a sample, for example, comprising anucleic acid molecule and/or reagents for performing a nucleic acidamplification reaction. A second dispersed phase 305 comprises adecontamination fluid. In some instances, the second dispersed phase isinjected. In some instances, the second dispersed phase is sampled butnot injected. A third dispersed phase 307 comprises a separation fluid.A fourth dispersed phase comprises a purge fluid 309. The separationfluid may be immiscible with the first, second, or third dispersedphase. In some instances, the separation fluid is injected following thepurge fluid. In some instances, the separation fluid is injectedfollowing the second dispersed phase. In some instances, the separationfluid is injected following the second dispersed phase and following thethird dispersed phase. In some instances, the separation fluid isinjected prior to the injection of the first dispersed phase. Theseparation fluid may be injected in interspersed amounts. The firstdispersed phase 303 comprising samples, a second dispersed phase 305, athird dispersed phase 307, and a fourth dispersed phase 309 pass to asampling device (“sampler”) 309 and then an injector 313. A continuousphase from a continuous phase reservoir 315 may be added to the injector313. From the injector 313, first dispersed phase 303 comprisingsamples, a second dispersed phase 305, a third dispersed phase 307, anda fourth dispersed phase 309 pass to a reactor 317 and a detector 319.

Separation (spacer) fluids as used in systems and method as describedherein may be used between dispersed phases. In some instances, theseparation fluid is immiscible with the dispersed phases. In someinstances, the separation fluid does not form a plug in a channel ortube. In some instances, the separation fluid comprises a size that doesnot fill a cross section of the channel or the tube. In some instances,the separation fluid comprises a flat velocity profile. In someinstances, the separation fluid has a greater affinity or surface energyfor a surface of the channel or the tube than a dispersed phase. In someinstances, the separation fluid has a lower affinity for a surface ofthe channel or the tube than a dispersed phase. In some instances, theseparation fluid comprises an oil. In some instances, a movement of theseparation fluid is in laminar flow. In some instances, the velocityfront is curved.

Systems and methods as described herein may comprise a plurality ofdispersed phases, wherein each dispersed phase of the plurality ofdispersed phases is immiscible with another dispersed phase. In someinstances, the dispersed phase is miscible with another dispersed phase.In some instances, due to the immiscibility of each of the dispersedphases, serial injection of the plurality of dispersed phases does notresult in cross-contamination.

A volume of the dispersed phase may be at least or about 0.001 nanoliter(nL), 0.002 nL, 0.003 nL, 0.004 nL, 0.005 nL, 0.006 nL, 0.007 nL, 0.008nL, 0.009 nL, 0.01 nL, 0.02 nL, 0.03 nL, 0.04 nL, 0.05 nL, 0.06 nL, 0.07nL, 0.08 nL, 0.09 nL, 0.10 nL, 0.20 nL, 0.30 nL, 0.40 nL, 0.50 nL, 0.60nL, 0.70 nL, 0.80 nL, 0.90 nL, 1.0 nL, 2.0 nL, 3.0 nL, 4.0 nL, 5.0 nL,10.0 nL, 20 nL, 30 nL, 40 nL, 50 nL, 60 nL, 70 nL, 80 nL, 90 nL, 100 nL,or more than 100 nL. In some instances, the volume of the dispersedphase comprises at least or about 100 nL, 200 nL, 300 nL, 400 nL, 500nL, 600 nL, 700 nL, 800 nL, 900 nL, 1000 nL, 2000 nL, 3000 nL, 4000 nL,5000 nL, 6000 nL, 7000 nL, 8000 nL, 9000 nL, 10000 nL, 20000 nL, 30000nL, 40000 nL, 50000 nL, 60000 nL, or more than 60000 nL. In someinstances, volumes of the dispersed phase injected is at least or about0.001 nanoliter (nL), 0.002 nL, 0.003 nL, 0.004 nL, 0.005 nL, 0.006 nL,0.007 nL, 0.008 nL, 0.009 nL, 0.01 nL, 0.02 nL, 0.03 nL, 0.04 nL, 0.05nL, 0.06 nL, 0.07 nL, 0.08 nL, 0.09 nL, 0.10 nL, 0.20 nL, 0.30 nL, 0.40nL, 0.50 nL, 0.60 nL, 0.70 nL, 0.80 nL, 0.90 nL, 1.0 nL, 2.0 nL, 3.0 nL,4.0 nL, 5.0 nL, 10.0 nL, 20 nL, 30 nL, 40 nL, 50 nL, 60 nL, 70 nL, 80nL, 90 nL, 100 nL, or more than 100 nL. In some instances, dispersedphase is injected into the process system (such as sample and/or spacerfluid); volumes of the dispersed phase injected are in a range of about0.1 uL to 100 uL, or 0.5 uL to 1000 uL, or 1 uL-200 uL, or 1 uL-100 uL,or 1 uL-70 uL, or 1 uL to 50 uL, or 5 uL-200 uL, or 10 uL-200 uL, or 10uL-100 uL, such as 0.1 uL to 100 uL, for example 0.5 uL to 80 ul, suchas 1 uL to 40 uL. In some instances, volumes of the dispersed phaseinjected is at least or about at least or about 100 nL, 200 nL, 300 nL,400 nL, 500 nL, 600 nL, 700 nL, 800 nL, 900 nL, 1000 nL, 2000 nL, 3000nL, 4000 nL, 5000 nL, 6000 nL, 7000 nL, 8000 nL, 9000 nL, 10000 nL,20000 nL, 30000 nL, 40000 nL, 50000 nL, 60000 nL, or more than 60000 nL.Systems and methods described herein for serial flow emulsion reactionsmay comprise volumes of dispersed phases that are further partitionedinto droplets. In some instances, the dispersed phase comprises sampleassays or discrete reactions. In some instances, volumes of dispersedphases are separated. In some instances, droplets (partitions) of thedispersed phases are separated. In some instances, volumes of dispersedphase partitions (e.g. droplets), e.g., after dispersed phase, such as asample, has moved through a partitioner, is at least or about 0.001nanoliter (nL), 0.002 nL, 0.003 nL, 0.004 nL, 0.005 nL, 0.006 nL, 0.007nL, 0.008 nL, 0.009 nL, 0.01 nL, 0.02 nL, 0.03 nL, 0.04 nL, 0.05 nL,0.06 nL, 0.07 nL, 0.08 nL, 0.09 nL, 0.10 nL, 0.20 nL, 0.30 nL, 0.40 nL,0.50 nL, 0.60 nL, 0.70 nL, 0.80 nL, 0.90 nL, 1.0 nL, 2.0 nL, 3.0 nL, 4.0nL, 5.0 nL, 10.0 nL, 20 nL, 30 nL, 40 nL, 50 nL, 60 nL, 70 nL, 80 nL, 90nL, 100 nL, or more than 100 nL. In some instances, volumes of dispersedphase partitions comprises at least or about 100 nL, 200 nL, 300 nL, 400nL, 500 nL, 600 nL, 700 nL, 800 nL, 900 nL, 1000 nL, 2000 nL, 3000 nL,4000 nL, 5000 nL, 6000 nL, 7000 nL, 8000 nL, 9000 nL, 10000 nL, 20000nL, 30000 nL, 40000 nL, 50000 nL, 60000 nL, or more than 60000 nL. Insome instances, volumes of dispersed phase partitions are in a range ofabout 10 pL to about 2 nL.

Provided herein, in some embodiments, are a series of sampler intakeimmersion stations for decontamination and/or purging of the fluidsampler intake. The sampler intake immersion stations may comprise awell, a valve, a reservoir of decontamination fluid, separation fluid,or purging fluid, a controller, and a flow pathway between the reservoirof decontamination fluid and/or separation fluid and/or purging fluidand the well. The sampler intake may enter a well and causes a valve tobe partially or wholly opened for partially filling the well withdecontamination fluid or purging fluid. The decontamination or purgingfluid may contact both an outer portion of the sampler intake and aninner portion of the sampler intake. A controller may cause the injectorto inject a portion of the decontamination fluid or purging fluid intothe fluid pathway between the sampler intake and the injector. In someinstances, the well is an open container for the contained fluid. Insome instances, the well has a cover that comprises an access port sothat contaminating elements may not enter the well but the samplerintake may have access. In some instances, the controller causes thesampler intake to draw a portion of the decontamination fluid or purgingfluid into the injection device. An amount decontamination fluid orpurging fluid that may be injected may comprise at least or about 0.001nanoliter (nL), 0.002 nL, 0.003 nL, 0.004 nL, 0.005 nL, 0.006 nL, 0.007nL, 0.008 nL, 0.009 nL, 0.01 nL, 0.02 nL, 0.03 nL, 0.04 nL, 0.05 nL,0.06 nL, 0.07 nL, 0.08 nL, 0.09 nL, 0.10 nL, 0.20 nL, 0.30 nL, 0.40 nL,0.50 nL, 0.60 nL, 0.70 nL, 0.80 nL, 0.90 nL, 1.0 nL, 2.0 nL, 3.0 nL, 4.0nL, 5.0 nL, 10.0 nL, 20 nL, 30 nL, 40 nL, 50 nL, 60 nL, 70 nL, 80 nL, 90nL, 100 nL, or more than 100 nL. In some instances, the amount ofdecontamination fluid or purging fluid that is drawn comprises at leastor about 100 nL, 200 nL, 300 nL, 400 nL, 500 nL, 600 nL, 700 nL, 800 nL,900 nL, 1000 nL, 2000 nL, 3000 nL, 4000 nL, 5000 nL, 6000 nL, 7000 nL,8000 nL, 9000 nL, 10000 nL, 20000 nL, 30000 nL, 40000 nL, 50000 nL,60000 nL, or more than 60000 nL. After drawing a volume of thedecontamination fluid, separation fluid, or purging fluid into theinjector, the controller may cause the sampler intake to stop drawingdecontamination fluid, separation fluid, or purging fluid into thepathway between the sampler intake and the injector. In some instances,the controller causes the injector to stop injecting decontaminationfluid or purging fluid to a waste channel or a flow pathway. Theinjector may leave the reservoir, which causes the valve to be closed.In some instances, the well comprises a volume of decontamination fluidand/or purging fluid. In some instances, the well comprises at least orabout 0.10 nL, 0.20 nL, 0.30 nL, 0.40 nL, 0.50 nL, 0.60 nL, 0.70 nL,0.80 nL, 0.90 nL, 1.0 nL, 2.0 nL, 3.0 nL, 4.0 nL, 5.0 nL, 10.0 nL, 20nL, 30 nL, 40 nL, 50 nL, 60 nL, 70 nL, 80 nL, 90 nL, 100 nL, or morethan 100 nL. In some instances, the reservoir comprises at least orabout 100 nL, 200 nL, 300 nL, 400 nL, 500 nL, 600 nL, 700 nL, 800 nL,900 nL, 1000 nL, 2000 nL, 3000 nL, 4000 nL, 5000 nL, 6000 nL, 7000 nL,8000 nL, 9000 nL, 10000 nL, 20000 nL, 30000 nL, 40000 nL, 50000 nL,60000 nL, or more than 60000 nL. In some instances, the well furthercomprises a drain to allow at least some of the decontamination fluidand/or purging fluid to leave the well. In some instances, the drainfurther comprises a valve that opens when the fluid injector is not inthe valve and closes when the fluid injector is in the valve. In someinstances, the decontamination fluid comprises water. In some instances,the decontamination fluid comprises sodium hypochlorite, phosphoricacid, sodium hydroxide, RNAse, or DNAase. In some instances, thedecontamination fluid comprises at least or about 0.5%, 1.0%, 1.5%,2.0%, 4.0%, 6.0%, 8.0%, 10%, 12%, 14%, 16%, 18%, 20%, 24%, 28%, 32%,34%, 36%, 40%, 44%, 50%, 60%, 70%, 80%, 90%, or more than 90% of sodiumhypochlorite, phosphoric acid, sodium hydroxide, RNAse, or DNAse, or acombination thereof.

Thus, a sampling device for use in systems and methods described hereinmay comprise a first dispersed phase reservoir, a second dispersed phasereservoir, a valve to select between the two reservoirs, and a tubeconnecting the various reservoirs to the valve and the valve to theinjector. In some instances, a filter is added in between the firstdispersed phase reservoir and the valve or between the valve and theinjector. The pump may be placed either upstream of the injector inletor downstream on an injector waste line. In some instances, the pump isplaced downstream on an injector waste line. The valve may allow thefirst dispersed phase to fill the injector and then switches to onlyallow second dispersed phase to fill the injector. In some instances,the sampling device comprises a reservoir of dispersed phases comprisingdecontamination fluid, purge fluid, or separation fluid that isconfigured to quickly disconnect. In some instances, the sampling devicecomprises a valve that is configured to be flushed with second dispersedphase. Multiple reservoirs of dispersed phases comprising samples orassays and/or dispersed phases comprising decontamination fluid, purgefluid, or separation fluid may be used.

Multiple dispersed phases may be introduced into the system. In someinstances, the dispersed phase is different from a dispersed phasecomprising sample. In some instances, the multiple dispersed phases areprovided from a common reservoir that is sampled after each dispersedphase comprising sample is injected. The reservoirs may be opencontainers of the multiple dispersed phases. The reservoirs may befitted with a cover with a central poppet valve. In some instances, whenthe sampling tube and/or lance contacts the poppet, the valve depressesand provides access to the contents of the reservoir. In some instances,the sampling tube and/or lance are immersed in the reservoir. If thefluid is decontamination fluid, both the exterior and interior surfacesof the lance/sampling tube may be decontaminated. The reservoir may havea cover, a central poppet valve, an external reservoir, and a tubeconnecting the reservoir and external reservoir. When depressed, thecentral poppet valve may provide access to the contents of the reservoirand opens a pathway that releases a fixed volume of fluid from theexternal reservoir into the reservoir. The external reservoir maycontinuously replenish the reservoir at each injection. Because thecentral poppet valve may isolate the external reservoir and thereservoir when closed, the central poppet valve may allow for theexternal reservoir to be exchanged and/or refilled in betweeninjections.

In some instances, the sampling system and the reservoir system are usedin conjunction. In some instances, the central poppet is depressed asthe lance is actuated downward. In some instances, the vertical stoprests on a top surface of the reservoir cover and the first springallows for the central tube to continue to move generally downward intothe reservoir. In some instances, when fully extended, both the exteriorand interior surfaces of the lance and the sampling tube are immersed inthe reservoir fluid at least to the level at which they are immersedwhen sampling first dispersed phase. In some instances, the samplingsystem pulls the reservoir fluid into the sampling tube with the pumpand then the sampling system retracts first the tube then the lance.

Barriers

In the system, samples may have small volumes (<1 mL, often <50 uL) andare placed into a set of containers (also referred to herein as astaging component, or similar wording) (e.g. wells of a microtiterplate) that is sampled over time (in some cases over 30 min, up to over6 hr or more). Such a small volume will be susceptible to evaporation.Thus, sealing these with a vapor barrier can prevent such evaporationand allow as much of the sample to be tested as possible while reducingerrors in concentration measurements. Additionally, foreign material inthe ambient environment surrounding the containers can be prevented fromentering the containers and potentially contaminating the sample if abarrier is in place.

One such vapor barrier can be a film placed over the top of each samplecontainer. In certain embodiments, this barrier comprises an adhesivefilm. The film can, e.g., cover more than one of the sample containers.In certain embodiments, the film covers all of the sample containers ofa set of sample containers, e.g., a microtiter plate. Any suitablematerial may be used for the seal; potential materials of constructionare a metal film, such as a film comprising aluminum, steel, copper, orany other metal, a polymer film, such as a film comprising polyethylene,acrylic, acrylonitrile butandiene styrene, bioplastics, cellophane,cellulose acetate, fluorinated polymers (PTFE, PVDF, ECTFE, FEP, PFA),nylon, polyamide acetal, polybutylene terephthalate, polycarbonate,polyester, polyetheretherketone, polyethersulfone, polyetherimide,polyethylene, polyimide, polyamide imide, polymethylpentene,polyolephins, polypropylene, polysulfone, polyphenylene sulfide,polyvinyl chloride, thermoplastic polyurethane], an elastomeric film,such as a film comprising silicone, rubber, hexafluoropropylene,ethylene propylene diene terpolymer, vinylidene fluoride,tetrafluoroethylene, vinylidene fluoride, hexafluoropropylene,perfluoromethylvinylether, acrylonitrile, polydimethylsiloxane,fluorosilicone, polyisoprene, polyisobutylene, polychloroprene,fluoroelastomers, polyurethane, epichlorohydrin, perfluoroelastomer,polysulfide, polytetrafluoroethylene, styrene butadiene,tetrafluoroethylene, ethylene acrylic, or any combination thereof.Unlike in typical real-time PCR machines (that also use a film to coverthe sample containers and prevent evaporation), the film does not needto be either transparent or temperature stable above 44° C. The film maybe placed over the container or containers by any suitable method, e.g.,by hand, by roller, or by using a thermal plate sealer. Important tothis method can be a system to pierce the seal prior to sampling. Anysuitable system and method may be used; exemplary systems and methodsare described herein. The film may be pre-perforated so as to breakrepeatably at the same position without fracturing into pieces smallerthan the effective inlet size of the inlet region of the intake channel.The film may instead comprise a resealable polymer material, so thatafter sampling, the system can again prevent evaporation and/orcontamination. This is useful should it be desirable to measure a singlesample multiple times. An added advantage of such an approach is that itprovides a “wiping” surface to remove fluids and/or solid materialsadhering to the sampling tip, reducing the amount of sample materialthat may remain on the tip and helping to prevent cross-contamination.

Thus, the dispersed phase reservoir (set of containers) comprising areaction sample may comprise a seal to reduce or prevent evaporation orto reduce or prevent contamination. In some instances, the seal adheresto a top surface of the first dispersed phase reservoir. In someinstances, the seal is broken or removed without contaminating thecontents of the first dispersed phase reservoir. In some instances, theseal is resistant to temperatures above about 20° C. to about 40° C. Insome instances, the seal is not resistant to temperatures above about20° C. to about 40° C. In some instances, the seal is transparent. Insome instances, the seal is not transparent. In some instances, the sealis opaque. In some instances, a reservoir aspect ratio is chosen suchthat a material comprising the seal will not contact the surface of thefluid contents of the first dispersed phase reservoir when broken. Insome instances, the seal comprises material of sufficient strength suchthat the seal will not fragment when broken or torn. The seal maycomprise an adhesive tape or film. In some instances, the seal comprisesa metal with an adhesive on one side of the seal. In some instances, theseal comprises a polymer material. Exemplary polymer materials include,but are not limited to, polyethylene, high density polyethylene, lowdensity polyethylene, polypropylene, or combinations thereof.

Figures as described below illustrate a sampler intake, wherein thesampler intake comprises a sharp, hard tube (the “lance”) for breakingthrough the seal and a sampling tube. In some instances, a knife, aneedle, or any pointed device that forms a wedge at the surface of theseal and ruptures the seal is used. The sampling tube may besubstantially concentric with the lance and resides inside the lance.The lance assembly may be mounted using a double spring comprising afirst spring and a second spring for compliance and a vertical stop. Asthe lance is actuated toward the seal, the lance may push through theseal until the vertical stop contacts the surface of the stagingcontainer (e.g. a microwell plate, or a PCR strip or tube, or an elementof a component designed to hold such a plate, PCR strip, or PCR tube).The vertical stop may prevent the lance from moving further, but thefirst spring allows for the sampling tube to continue traveling outwardwithin the lance. The inlet of the sample tube is actuated until itreaches a position in the contents of the first dispersed phasereservoir. In some instances, the sampling tube continues to move untilit contacts a bottom of the first dispersed phase reservoir. In someinstances, the second spring allows the sampling tube to continue tomove in the direction of first dispersed phase reservoir. In someinstances, the second spring allows the sampler intake to self-positionin the bottom of the tube. In some instances, the first and secondspring provide x-y compliance as well as z-compliance.

The sampling system may prevent contamination of the inner sampling tubeby the seal because the sampling tube is substantially contained withinthe lance as the lance breaks the seal. In some instances, the lancepushes the seal material such that the seal material is prevented fromcontacting the tube surface. In some instances, the sampling tube isattached to the lance. Once sample has been drawn out of the firstdispersed phase reservoir or any dispersed phase reservoir, the samplingtube may be retracted inside the lance and the assembly retracted byreversing the initial steps.

Another such vapor barrier is a layer of fluid comprising a fluid oflower density than the fluid to be sampled. In certain embodiments, thisfluid has a lower vapor pressure than the fluid to be sampled, althoughif a large enough volume of it is used, it can have a similar or highervapor pressure than the sample fluid. A metric is that the combinationof vapor pressure and volume added is such that the entire vapor barrierdoes not evaporate in the elapsed time before the fluid would besampled. An advantage of this approach is that the vapor barrier fluiddoes not need to be pierced or broken by the sampling head; not onlydoes this reduce the possibility of pieces of the vapor barrier sealbeing incorporated into the fluid being sampled, but it allows the fluidbeing sampled to “reseal” once the sampling head leaves the well. If thevapor barrier fluid has a higher viscosity than the fluid being sampledand/or a higher affinity for a material comprising the surface of thesampling head than does the fluid being sampled, the vapor barrier canact to “wipe” the sampling head clean of the fluid being sampled,leaving it in the container (e.g. a microtiter plate well). This helpsin preventing cross-contamination. In certain embodiments, the vaporbarrier fluid is added by the user before placing the sample containersinto the area that can be accessed by the sampling head (i.e. in theinstrument). In certain embodiments, the instrument is configured todispense vapor barrier fluid into the sample containers after the userplaces it into the area that can be accessed by the sampling head. Thisfluid can be dispensed through the same channel that sample fluid ispulled into the system, or it can be dispensed through at least oneseparate channel, for example as described above for the systemdispensing the dead volume fluid.

In certain embodiments, both a film seal and a vapor barrier fluid areused. In such an embodiment, the contents of a sample container comprisea sample fluid and the sample container is sealed by the film seal. Thecontainer is then positioned in the instrument and, at a subsequenttime, is pierced by the sampling head. The instrument can then add vaporbarrier fluid through a discrete channel. Such an approach eliminatesthe need for the user to handle the vapor barrier fluid, but reduces theamount of sample fluid that evaporates before the system can add vaporbarrier fluid.

In certain embodiments, the vapor barrier fluid and the dead volumefluid are both simultaneously in the sample container (e.g. a well in amicrotiter plate). As described above, these can be added by the systemor by the user (by hand or by a liquid handling robot), or anycombination of these.

This embodiment is referred to here as the “parfait” approach, as thedifferent phases (e.g., sample fluid, dead volume fluid, and vaporbarrier fluid) arrange themselves into a vertically striated system withclear phase boundaries. In a parfait, the vapor barrier fluid naturallyhas the lowest mass density; the mass densities of the sample and thedead volume fluid can be either greater or lower, respectively, from theother (but not the same). In some instances, the dead volume fluid andthe vapor barrier fluid have the same composition. Parfait embodimentsare described in more detail in the Figure descriptions, below.

Thus, a sampling device may be used for sampling from a well in a sealedplate. The sampling device may comprise a lance, sampling tube, firstspring, positive stop, second spring, actuation mechanism, and motor.The sampling device may interface with a sample that is sealed using aseal. The sampling device may be positioned vertically above the samplecontainer. The actuation mechanism may translate rotational motion ofthe motor into linear vertical motion of the lance, sampling tube firstspring, second spring, and positive stop. To sample the contents of thesample container, the motor and actuation mechanism together may beginto move the lance, sampling tube, first spring, second spring, andpositive stop toward the outer surface of the seal on the samplecontainer. Upon contact with the seal, the lance may pierce the seal andforces the seal material out of the path of the sampling tube so thatthe outside of the sampling tube does not contact the portion of theseal material that was not exposed to the interior of the samplecontainer. The motor and actuation mechanism may continue to move theassembly downward until the positive stop contacts a top surface of thesample container or a holder for the sample container. The positive stopmay restrict further movement of the lance and first spring. Continuedlinear actuation of the motor and actuation mechanism may cause thesampling tube to move downward within the lance. The sampling tube maycontinue to move downward until the actuation mechanism encounters asecond stop condition. In some instances, the stop condition is a totalelapsed time for motor actuation at a given set of rotational rates. Insome instances, the stop condition is a limit switch triggered by thevertical position of a portion of the assembly. In some instances, thelimit switch comprises an optical interrupter, a mechanical switch, amagnetic switch, or a combination thereof. In some instances, the stopcondition is an increase in motor current caused by an increase inresistance to motion caused by the sampling tube contacting the bottomof the sample container. In some instances, the motor is a stepper motoror a servo motor. In some instances, the stop condition is theachievement of a minimum number of steps. In some instances, the stopcondition is set such that the vertical position of the assembly and thebottom of the sample container will interfere so as to ensure that thesampling tube is at the bottom of the sample container. In someinstances, a negative pressure is created in a downstream portion of thesample tube so that a volume of fluid is pulled from the samplecontainer into the sampling tube. In some instances, negative pressureis generated using a pump. In some instances, negative pressure isgenerated using a peristaltic pump.

The second spring for use in sampling devices as described herein mayprovide compliance. In some instances, compliance allows for nosignificant impact to the motor or actuation mechanism if the stopcondition is encountered after the vertical positions of the samplingtube and the bottom of the sample container. In some instances, thesecond spring allows for flexible vertical or horizontal positioning.

Dispensing

In embodiments where at least one of the vapor barrier fluid and thedead volume fluid may be added by the instrument, the sampling head maycarry elements that allow for this dispensing. In certain embodiments,the intake channel itself allows for dispensing of the vapor barrierfluid, the dead volume fluid, or both. As an example, the samplingsystem may comprise the intake channel, the injector, a reversible pump,a selector valve, a waste channel, and a reservoir of either dead volumefluid or vapor barrier fluid. When dispensing fluid, the selector valveis positioned so as to connect the reservoir through the injector to theintake channel. The pump provides motive force so as to dispense fluidfrom the reservoir through the inlet end of the intake channel(effectively making it an outlet). When sampling, the selector valve ispositioned so as to connect the intake channel through the injector tothe waste channel. When sampling, the pump provides motive force to pullfluid from the sample container through the inlet of the intake channel,through the injector, and to waste. In certain embodiments, the pump isa peristaltic pump and is inline with the injector and sampling valve.In another embodiment, the system further comprises a syringe pump, aservice loop, and a second selector valve, and dispensing or injectingfirst involves pulling fluid into the service loop of the syringe pump,followed by pushing fluid out of the service loop of the syringe pump.In further embodiments, the system comprises yet another selector valveand a second reservoir of fluid (whichever fluid was not alreadyincluded).

In certain embodiments, the dispensing channel is distinct from theintake channel. In certain embodiments, this channel is physicallyattached to the intake channel, so that positioning the intake channelof the injector effectively positions the dispensing channel. Doing sosaves the need for an independent positioning mechanism. In certainembodiments, the dispensing channel has its own mechanism forpositioning. In embodiments where the dispensing channel is distinct,dispensing of the fluid can be achieved by any suitable method. Incertain embodiments, the system comprises a pump, the dispensingchannel, and a reservoir of either dead volume fluid or vapor barrierfluid. Dispensing is achieved by actuating the pump to drive fluidthrough the outlet of the dispensing channel. In certain embodiments,the system comprises a selector valve and a second reservoir, andselection of fluids can be achieved by positioning the selector valve toconnect one of the reservoirs to the outlet of the dispensing channel.Any suitable pump may be used, such as a peristaltic pump, diaphragmpump, syringe pump, and the like. In certain embodiments, the pump is adiaphragm pump. In certain embodiments, the pump is a syringe pump andthe system comprises a second selector valve and a service loop, wherefluid is first pulled into the service loop before dispensing it via thesyringe pump.

In some embodiments, the sample container is not sealed before fluidsare dispensed by the system into it. In other embodiments, there is aseal on the sample container, and the system first breaks the sealbefore dispensing fluids into the system.

Tips

The systems and methods of bringing sample into the system can beimportant. The inlet region of the intake channel of the sample headmust be able to reliably convey sample fluid, dead volume fluid, orpotentially vapor barrier fluid into the system, and/or other fluid.Because the system comprises microfluidic channels, it is important thatparticulate matter that could partially or completely obstructmicrofluidic channels be rejected from the intake channel. It is notalways possible to control the composition of the fluids to be drawninto the intake channel (especially in the case of sample fluid, whichis provided by a user), and so systems and methods to prevent intake ofproblematic particulate matter are important. Additionally, in systemsthat have film-based seals over the sample container, methods topuncture or pierce that seal without contaminating the sample orintroducing problematic particulate matter can be provided. Finally, thematerials of construction of the elements of the sampling head that comeinto contact with the working fluids of the system can be such that theydo not promote (and preferably, they hinder) cross-contamination in thesystem.

In certain embodiments, the inlet portion of the intake channel issimply a tube. Any suitable material of construction can be used.Exemplary materials of construction include polymers, fluoropolymers,glass, stainless steel, carbon steel, aluminum, titanium, or acombination thereof, or other suitable materials. In the case that amaterial of the tube comprises a material that has a substantialaffinity for the fluid to be sampled or contaminating material such asdetectable (or potentially detectable) material, the tube can have acoating or lining that comprises a material that does not have asubstantial affinity for aqueous phase or contaminating material such asdetectable (or potentially detectable) materials. In certain embodimentsthe surface comprises a material that has a greater affinity forhydrophobic or fluorophilic substances. In an example, the coating is afluoropolymer. Rejection of problematic particulate material can beachieved by choosing a major channel dimension that is smaller than thesmallest representative dimension of the particulate material to berejected. For example, if the tube has a circular cross section, itsdiameter should be chosen so that it is smaller than a representativediameter of the particulate matter to be rejected. Such a tube can havea circular, oval, square, or any other suitable cross section. In anexample, the tube is a continuous fluoropolymer tube that changesdiameter between the inlet region and the injector so that particulaterejection can be achieved but flow is not overly restricted.Representative diameters in the inlet region are typically in the range40 microns to 200 microns and in the region beyond the inlet region aretypically in the range 50 microns to 5 mm. In another example, the tubeis the interior of a stiff metal or polymer needle. The needle surfacescan be coated with a polymer, fluoropolymer or the like to preventadherence of aqueous phase or detectable (or potentially detectable)material. The needle diameter may vary across its length, but at theinlet end it should be small enough to reject unwanted particulatematter. In some examples, the inlet end diameter ranges between 40microns and 110 microns. In certain embodiments, the needle is removableand replaceable, so that should it become clogged, it can be replacedwithout replacing the system.

In certain embodiments, the inlet end of the intake channel comprises amicromachined channel, a representative dimension of which is smallenough that the smallest unwanted particulate material cannot pass intothe intake channel. In an example, the micromachined channel comprises ahole in a substrate, and the substrate can be connected to a tube orchannel comprising the proximate portion of the intake channel. Inexamples, the substrate may be disconnected and replaced to allow formaintenance (e.g. if the micromachined channel becomes fouled orcontaminated). The micromachined channel can be made of any suitablematerial, e.g., the same materials as described for the tube above.Micromachining may be done by any suitable operation, includingdrilling, milling, and/or laser drilling. In certain embodiments, theinlet end of the intake channel may be formed by a method comprisingheating a polymer tube, applying a tensile force to the tube so as toreduce the cross-sectional area of the tube in a region, and cutting atube in the region to form the inlet end of the intake channel. Incertain embodiments, the inlet end of the intake channel may be formedby a method comprising inserting a mandrel of a desired cross-sectionalarea or profile into the internal volume of a polymer tube, applying acompressive force to the polymer tube so as to reduce thecross-sectional area of the tube in a region around the mandrel to thatof (or nearly of) the mandrel, and cutting the tube in the region. Incertain embodiments, the method additionally comprises heating the tube.

In certain embodiments, the inlet end of the intake channel comprises abundle of tubes meeting at a common junction. Each of the tubes in thebundle has a characteristic dimension, e.g., diameter that is smallerthan a characteristic dimension, e.g., diameter, required to rejectproblematic particulate material from the system. Beyond the junction,the intake channel of the instrument comprises the common channel thatjoins all the tubes in the bundle.

Thus, in some instances systems and methods of the invention provide afilter. The filter may be used to remove particulate matter that mayblock a flow in the injection device, droplet generator, microfluidicchannel, PCR reactor, or detection device. In some instances,particulate matter is a result of variation in a composition ofbiological samples, incomplete digestion and/or lysis of cellularcomponents during sample preparation, introduction of foreign materialdue to user error or laboratory environmental conditions, variances inmanufacturing tolerances, and/or cleanliness, in the individualcomponents comprising the sampling device, or any other source. In someinstances, the filter comprises a single channel of a first dimension oflower value than a second dimension of the particulate matter. The firstdimension may be of a dimension such that particulate matter comprisingthe second dimension is prevented from being blocked by the filter. Insome instances, the first dimension is a hydraulic diameter, across-sectional area, or a circular diameter. In some instances, thesecond dimension is a Feret's Diameter, a Martin's diameter, an aspectratio, projected area diameter, or dynamic diameter.

In some instances, the single channel comprises a polymer tube. In someinstances, the polymer tube comprises a portion of its length where atube diameter has been constricted. For example, the tube diameter isconstricted by applying a tensile force along the tube. In someinstances, the tube diameter is constricted by heating the tube andapplying a tensile force along the tube. In some instances, a ratio ofthe smallest tube diameter to the bulk tube diameter is at least orabout 0.2:1, 0.25:1, 0.5:1, 0.7:1 0.75:1, 1:1, 1.25:1, 1.5:1, 1.75:1,2:1, 2.25:1, 2.5:1, 2.75:1, 3:1, 3.25:1, 3.5:1, 3.75:1, 4:1, 4.25:1,4.5:1, 4.75:1, 5:1, 5.25:1, 5.5:1, 5.75:1, or 6:1. The ratio of thesmallest tube diameter to the bulk tube diameter is a ratio as toprevent pinching or mechanical failure of the tube. In some instances, aratio of the tube length in the constricted section to the total tubelength is at least or about 0.2:1, 0.25:1, 0.5:1, 0.7:1 0.75:1, 1:1,1.25:1, 1.5:1, 1.75:1, 2:1, 2.25:1, 2.5:1, 2.75:1, 3:1, 3.25:1, 3.5:1,3.75:1, 4:1, 4.25:1, 4.5:1, 4.75:1, 5:1, 5.25:1, 5.5:1, 5.75:1, or 6:1.

The single channel may comprise one or more tubes of varying sizes. Forexample, the single channel may comprise a tube of a first size measurehaving a restriction comprising a second, smaller size measure placed atsome point along the tube. In some instances, the first size is ahydraulic or circular diameter. In some instances, the second sizemeasure is a hydraulic or circular diameter. The restriction may be heldin the system by a housing. In some instances, a volume of fluid in thehousing prevents fluid holdup. In some instances, a shape of therestriction is rectangular, tubular, oblong, circular, round, oval. Insome instances, a shape of the restriction is circular. In someinstances, a shape of the restriction is rectangular.

Thus, in some instances, a filter is used in the systems and methods asdescribed herein. In such an arrangement, a cross-sectional area of thepolymer input tubing is reduced such that the cross-sectional area issmaller than particulate matter that is blocked from entering. In someinstances, the cross-sectional area of the input tubing, e.g., polymerinput tubing is at least or about 1 to 100 um. In some instances, amicrofluidic chip with a channel that has a reduced cross-sectional areais used. The channel of the microfluidic chip may be constructed bypassing the channel through a constriction with an increase gain insize. In some instances, such a construction results in a filter that iscompact and easy to place in-line.

In some instances, multiple channels are used to restrict particulatematter from entering. The multiple channels may comprise a firstdimension smaller than a second dimension of the particulate matter. Insome instances, the first dimension blocks particulate matter. In someinstances, the first dimension allows fluid to pass. In some instances,a filter comprising porous material having pores of a first dimension isused to allow fluid to pass but block particulate matter. In someinstances, a sampling tube is a lumen designed to reject particulatematter before it enters the system. In some instances, a chip-basedsystem where a central channel branches to multiple channels forrestricting particulate matter that then rejoin a main channel is used.

In certain embodiments, the inlet end of the intake channel hassufficient stiffness to pierce a film seal, as described above. Inoperation, the inlet end of the intake channel is positioned over theseal, force is applied to push the tip through the seal, and the tipthen continues to travel into the sample. In some embodiments, the tipstops moving after piercing the seal, and then dispenses a fluid, suchas a dead volume fluid, vapor barrier fluid, or both, into the samplecontainer (e.g. a well in a microtiter plate). In certain embodiments,the sampling head comprises a separate device for piercing a film seal.Any suitable device may be used, such as a needle, a lancet, a lance, orany other appropriate means for piercing a seal.

Injector positioning. For a variety of reasons, it can be important toknow to some practical extent the composition of the materials in theinjector chamber, i.e., common conduit. First, it is desired to maximizethe amount of sample that enters the process system. If the sample isnot fully in the common conduit, then some amount of sample will berejected when the injector is positioned to inject the sample, e.g.,when the injector rotates. Second, it is important to prevent air fromentering the process system. At the conditions of operation of theprocess system, air is a compressible gas. It may expand and/or contractin a reactor that employs heating or cooling, e.g., a thermal cycler,which causes periodicity in flow in the system, which affects, e.g., PCRtiming. Additionally, it provides fluidic capacitance, which affectspartition generation.

Thus, it is advantageous to be able to determine when 1) sample is inthe injector chamber (common conduit) and/or 2) when air may be in theinjector chamber (common conduit). Various systems and methods toachieve these objectives are described here. One set of ways todetermine whether sample is in the injector is to use optical propertiesof the fluids in the injection system to determine which fluids arebeing added. In certain embodiments, at least one optical property ofthe sample fluid is sufficiently different from the at least one opticalproperty of the dead volume fluid and/or vapor barrier fluid that, inquantifying the optical property, the sample fluid may be distinguishedfrom the other fluids in the system. A detector capable of quantifyingthe optical property may be placed at a first distance upstream of theinjector, and the optical property may be measured at this point. Thevolume of the intake channel between the injector chamber (commonconduit) and the measurement point is a known function of the firstdistance, and the volume of the intake chamber (common conduit) isknown. At the point in time that sample fluid is first indicated by achange in the measurement of the optical property, the system may draw aspecific additional volume into the system related to the sum of thevolume of the injector chamber (common conduit) and the volume of intakechannel between the measurement point and the injector chamber (commonconduit). In the case that the sample is of a smaller volume than theinjector chamber (common conduit) and the user desires to contain theentire sample in the injector chamber (common conduit), this volume willbe at least as large as the sum of the volume of the sample fluid andthe volume of the intake line between the measurement point and theinjector chamber (common conduit). In the case that the user desires toinject only sample fluid into the process side of the system and thevolume of the sample fluid is larger than the volume of the injectorchamber (common conduit), the drawn volume will be at least as large asthe sum of the volumes of the injector chamber (common conduit) and theintake channel between the measurement point and the injector chamber(common conduit). In the case that the user wishes to avoid theinjection of a fluid upstream (“trailing”) of the sample fluid, thedrawn volume will be less than the sum of the volumes of the samplefluid and the intake channel between the measurement point and theinjector chamber (common conduit).

In certain embodiments, the optical property of the fluids is arefractive index and the change in fluids is detected by a change in theexit angle of a electromagnetic radiation source passing through theintake channel. Any suitable electromagnetic radiation source may beused, such as a light-emitting diode (LED), a laser, an incandescentlight, or any combination thereof. In certain embodiments, the opticalproperty is an absorbance or a scattering albedo in a wavelength rangeand the change in fluids is detected by the change in the intensity ofelectromagnetic radiation absorbed or scattered from the sample fluid(the electromagnetic radiation could either be detected in atransmissive or reflective setup). In certain embodiments, thewavelength range is in the infrared spectrum.

In certain embodiments, at least one of the fluids comprises a componentthat emits electromagnetic radiation in a first wavelength range whenexcited by a electromagnetic radiation source in a second wavelengthrange. In this case, the detector comprises a electromagnetic radiationsource and a photodetection element. Changes in the emitted intensity ofelectromagnetic radiation from the intake channel, when excited,indicate changes in the fluids passing through the sample. In anexample, the sample fluid comprises at least one excitable component(these could be the same or different as the fluorescent molecules usedto detect PCR products), and the dead volume fluid or vapor barrierfluid do not comprise the at least one excitable component. When theintensity of electromagnetic radiation detected in the wavelength rangeof emission for the at least one excitable component increases above aminimum value, the presence of the sample fluid in the intake channel atthe point of measurement is established. Likewise, when the intensity ofelectromagnetic radiation decreases below the minimum value, the absenceof the sample fluid in the intake channel at the point of measurement isestablished. In other examples, the at least one excitable molecule maybe in the dead volume fluid or in the vapor barrier fluid (or both), andthe absence of an intensity above the minimum value indicates presenceof the fluids (preferably the sample fluid) that do not contain the atleast one excitable component. In an embodiment, the at least oneexcitable component comprises a fluorescent molecule, e.g., as describedherein. In another embodiment, at least one excitable componentcomprises a phosphor or a quantum dot.

In certain embodiments, more than one of the fluids contain at least oneexcitable component, for example at least one pair of excitablecomponents whose emissive wavelength ranges do not substantiallyoverlap. In this embodiment, specific fluids in the system may beidentified by the wavelength of electromagnetic radiation emitted by thefluid under irradiation by at least one electromagnetic radiationsource. This is especially valuable if the volume of sample in thesample container is unknown, as the start and end of a sample may bedemarcated as it passes by the measurement point by measuring theelectromagnetic radiation intensity at the relevant wavelengths. Anexample of this in a PCR system is to put an oil soluble dye into thevapor barrier fluid, or whatever fluid is trailing the sample fluid, andusing a water soluble dye (or the PCR probes themselves) in the samplefluid to detect the sample. A minimum intensity at a first wavelength isdetected, indicating the start of a sample, and a minimum intensity at asecond wavelength is detected, indicating the start of the trailingfluid.

In certain embodiments, more than one of the fluids contain at least oneexcitable component, where at least one first fluid contains at leastone excitable component with an emissive wavelength range thatsubstantially overlaps with the emissive wavelength range of at leastone excitable component in at least one second fluid. In thisembodiment, specific fluids in the system may be identified by theintensity of the signal at the emissive wavelength range of thecomponent(s) with common emissive wavelength ranges. For this to besuccessful, the quantified emitted intensity from each fluid for a givenexcitation intensity should be different so that the two fluids may bedistinguished.

Examples of methods to accept a variable volume of sample into theinjector and/or to detect/prevent injection of air into the systemfollows. In a first example, there is only sample fluid in the samplefluid container. Prior to sample fluid intake, the intake channelbetween the inlet end of the intake channel and the outlet of theinjector channel does not contain sample fluid (it may contain deadvolume fluid, vapor barrier fluid, air, or anything else). A pump on theintake channel draws sample fluid from the sample fluid container intothe intake fluid channel until the detector measures a value sufficientto indicate the front edge of the sample fluid. A controller directs thepump to continue to draw sample fluid into the system until the frontedge of the sample fluid is a known volume beyond the outlet of theinjector chamber (common conduit). If the controller has not detected achange in the quantity of the optical property sufficient to indicate achange from the sample fluid to air at the measurement point, the samplefluid may be injected. If the controller has detected a change in thequantity of the optical property sufficient to indicate a change fromthe sample fluid to air at the measurement point but the volume of fluiddrawn into the system after that point in time is less than the volumeof the intake channel between the measurement point and the injectorchannel plus the known volume beyond the injector channel the sample wasdrawn to, the sample may be injected without injecting air. If thecontroller has detected a change in the quantity of the optical propertysufficient to indicate a change from the sample fluid to air at themeasurement point and the volume of fluid drawn into the system afterthat point in time is greater than or equal to the volume of the intakechannel between the measurement point and the injector channel, then thesample fluid drawn was of insufficient volume to fill the injectorchamber (common conduit) and the sample fluid can be rejected withoutinjecting it into the process side of the system, thus preventing, e.g.,entry of air into the process side.

In a second example, there is both sample fluid and dead volume fluid inthe sample container, and the sample fluid has a lower mass density thanthe dead volume fluid. The inlet of the intake channel is positioned sothat it is substantially in the dead volume fluid, and the pump isactuated so that dead volume fluid begins to fill the intake channel.When the level of the fluid in the sample container decreases so thatthe interface between the sample fluid and the dead volume fluid is atthe same height as the inlet of the intake channel, sample fluid willbegin to follow the dead volume fluid into the intake channel. When thedetector measures a change in a quantifiable property (e.g., in aelectromagnetic radiation intensity) that indicates an interface betweenthe dead volume fluid and the sample fluid, the pump then continues todraw a volume of fluid equal to the sum of the volume of the intakechannel between the detector and the injector, the internal volume ofthe injector chamber (common conduit), and any desired overfill of theinjector. If the detector has not subsequently indicated a phaseinterface between the sample fluid and either air or dead volume fluid,or if the detector has subsequently indicated a phase interface betweenthe sample fluid and either air or the dead volume fluid but theadditional volume drawn into the intake channel after this detection ofa phase interface is less than the volume of the intake channel betweenthe detector and the injector inlet, then no air has been included inthe injector chamber (common conduit) and the sample may be injectedinto the process side of the system. Otherwise, the sample can berejected.

In a third example, there is sample fluid, dead volume fluid, and athird immiscible fluid (which may be a vapor barrier fluid or may simplybe a fluid designed to separate aliquots of sample injected into theprocess side of the system, e.g., spacer fluid) in the sample container.The inlet end of the intake channel is positioned so that it is in orsubstantially in the dead volume fluid. The pump is then actuated,causing dead volume fluid to be drawn into the intake channel. When thelevel of the fluid in the sample container decreases so that theinterface between the sample fluid and the dead volume fluid is at thesame height as the inlet of the intake channel, sample fluid will beginto follow the dead volume fluid into the intake channel. Once the samplefluid is completely drawn into the system, the third immiscible fluidbegins to be drawn into the system. Similar to above, once the detectorregisters an interface between the sample fluid and the dead volumefluid, the pump continues to draw fluid into the system. If a secondphase interface is not detected before the injector chamber (commonconduit) is completely filled with fluid, then the injector can injectthe sample into the system, and the injection will solely be sample. Ifa second phase interface is detected before the injector chamber (commonconduit) is completely filled with fluid, the pump can continue to drawfluid in a quantity equal to the volume of the intake channel betweenthe detector and the inlet of the injector. In this way, the intakechannel will be filled with a mixture of sample fluid and the thirdimmiscible fluid.

Cleaning Stations and Routines An important aspect of certainembodiments of systems and methods provided herein is the ability to usethe intake channel for multiple sample injections withoutcross-contaminating the process side of the system. This allows for theuse of higher quality components (e.g. tighter tolerances, machined, notinjection molded, etc.) in the systems, because the cost of thosecomponents will be amortized over many sample fluid injections, as wellas removing the requirement that a removable or disposable consumableelement be incorporated into the system, which may produce poorerresults due to the requirement that a user make/break connections (whichcan lead to channel misalignment, sharp interfaces, or air bubbles, allof which can impact droplet formation and stability) and that thedisposable be low-cost (which leads to lower tolerance requirements,impacting quality and consistency of droplet formation). Multipleaspects provided herein aid in preventing cross-contamination. One suchset of aspects are systems and methods for cleaning the intake side ofthe system between injections into the process side of the system.

In some embodiments, the surfaces of the channels in the intake systemcomprise materials that have a higher affinity for a cleaning fluid thanfor the sample fluid, allowing the cleaning fluid to displace the samplefluid when the cleaning fluid flows through the intake channel.“Cleaning fluid,” as that term is used herein, includes any fluid thatis moved through an intake system, e.g., an intake system including aninjector, to remove and/or render inactive any sample or othercontaminant in an intake pathway; exemplary cleaning fluids includepurge fluids and denaturing fluids, as well as, in some cases, deadvolume fluids and/or vapor barrier fluids, as well as any other fluidsthat can move through the intake side, e.g., without being moved intothe process side, as described herein. By substantially displacing allof the sample fluid in at least the injector chamber (common conduit),and, in some cases, the entire intake channel, the cleaning fluid mayprevent injection of sample fluid from previous samples when workingwith new sample fluid. In some embodiments, multiple cleaning fluids areused. The cleaning fluids may be immiscible with the sample fluid ormiscible with the sample fluid, but in general, at least one of thecleaning fluids is immiscible with the sample fluid.

Systems and methods are described here to introduce the at least onecleaning fluid into the intake channel. In certain embodiments, the atleast one cleaning fluid is supplied in a separate container from thesample container. In order to access the cleaning fluid, the inlet endof the intake channel is positioned so that it is substantially in thecleaning fluid in the at least one cleaning fluid container and a pumpis actuated so that the cleaning fluid is drawn into the intake channel.In embodiments where there are multiple cleaning fluids, each cleaningfluid may be supplied in a separate cleaning fluid container, and thecleaning fluids are sequentially drawn into the system by positioningthe inlet end of the intake channel so that it is substantially in eachof the cleaning fluids in the order in which cleaning fluids are to bedrawn into the system and the pump actuated for a given volume or timein each cleaning fluid. In some embodiments, air may be drawn into theintake channel by the (at least one) pump in between at least one of thecleaning fluids and the sample fluid or between at least two of theindividual cleaning fluids. In a further embodiment, air is addedbetween all of the fluid aspirations into the system. Air may beaccessed by positioning the inlet end of the intake channel so that itis no longer substantially within a liquid volume.

The cleaning fluid containers may take any suitable form. In certainembodiments, at least one of the cleaning fluid containers is adisposable container. For example, at least one of the cleaning fluidcontainers could be a well of a microtiter plate, a PCR tube, a strip ofPCR tubes, a conical-bottom tube, an injection molded polymer containermeant to be disposed of after a set number of uses, or the like. In thisembodiment, at least one of the cleaning fluid containers may beinitially sealed by a polymer or metal film or closed by a cap to makeit easier to supply to end users. In the case that it is sealed by afilm, the system will have the capability of breaking the film (asdescribed above).

In certain embodiments, at least one of the cleaning fluid containers isa fixed reservoir on the instrument over which the inlet end of theintake channel may be positioned. As an example, at least one cleaningfluid container may be an open tray filled with the cleaning fluid. Asanother example the cleaning fluid container may be a partially closedcontainer with an access port for the inlet end of the intake channel.The access port may have a door or reversible seal to close off thecleaning fluid container when the inlet end of the intake channel is notpositioned inside. In an example, the door may simply be mounted on aspring-loaded hinge that may be pushed open by the sampling head(comprised of the inlet end of the intake channel) and that wouldautomatically close as the sampling head disengages. In another example,the door can be a spring loaded poppet or similar device that opens whendepressed by the sampling head, but closes when the sampling head iswithdrawn.

Cleaning fluid may be provided to the cleaning fluid containers in avariety of ways. In certain embodiments, the user manually adds a volumeof cleaning fluid to the cleaning fluid reservoir sufficient to processat least one sample fluid volume in the system prior to operating thesystem. In certain embodiments, at least one cleaning fluid container issupplied with cleaning fluid through a cleaning fluid supply channelfrom a cleaning fluid reservoir that may be filled or interchanged bythe user. In certain embodiments, the cleaning fluid flows from thecleaning fluid reservoir into the cleaning fluid container by gravity.In one example, the cleaning fluid reservoir is completely emptied uponloading by gravity into the cleaning fluid container (in this example,the cleaning fluid reservoir provides a convenient means for loading thecleaning fluid container). In another example, the cleaning fluidcontainer has a smaller operating volume than the operating volume ofthe cleaning fluid reservoir and the system comprises a mechanism tocontrol dispensing of cleaning fluids into the cleaning fluid reservoir.As one example, the cleaning fluid reservoir may be closed to theambient atmosphere and of fixed overall volume so that, as the cleaningreservoir empties of fluid, the level of fluid in the cleaning fluidcontainer is controlled by a balance of ambient atmospheric pressure onthe surface of the cleaning fluid in the cleaning fluid container andthe sum hydrostatic head of the cleaning fluid in the cleaning fluidreservoir and any air pressure in the cleaning fluid reservoir. Inanother example, a valve controls dispensing of the cleaning fluid fromthe cleaning fluid reservoir into the cleaning fluid container. Thisvalve can be controlled by a controller on the system, opening the valveafter at least one cleaning fluid intake cycles so as to dispense a setvolume of fluid into the cleaning fluid reservoir. This set volume offluid can be measured in any suitable manner, e.g., by the hydrostaticpressure in the cleaning fluid container or a level sensor in thecleaning fluid container, such as a float valve, capacitance level, orthe like, by opening the valve for a set amount of time correlated to acalibrated flow rate in the channel connecting the cleaning fluidreservoir to the cleaning fluid container, by measuring a flow rate inthe channel connecting the cleaning fluid reservoir to the cleaningfluid container and integrating that flow rate over time so as todetermine when the set volume as been dispensed, by measuring a changein level in the cleaning fluid reservoir indicating when a set volumehas been dispensed, or any other suitable manner. In another example,the valve system is constructed so as to only allow a set volume offluid into the cleaning fluid container each time the valve is actuated.In this example, each time the valve is actuated, a priming chamber isfilled with cleaning fluid. On the subsequent actuation, the primingchamber is dispensed into the cleaning fluid container.

In certain embodiments, the cleaning fluid is contained in the samplecontainer. Due to differences in mass density and miscibility, thecleaning fluid forms a separate layer in the fluid “parfait” and may beloaded into the intake channel by changing the level of the inlet end ofthe intake channel so that it is substantially in the cleaning fluidlayer, by drawing fluid into the inlet end of the intake channel so thatthe level of the interface of the cleaning fluid with the fluid intowhich the inlet end of the intake channel was originally positionedfalls to be a level with the inlet end of the intake channel, or both.In certain embodiments, a cleaning fluid is also the dead volume fluid,the vapor barrier fluid, or both. Cleaning fluids may be added to thesystem in this approach in the same way as for the “parfait” approachesdescribed above.

In certain embodiments, at least one cleaning fluid container comprisesat least one cleaning fluid supply line and one cleaning fluid drain. Atleast one cleaning fluid is supplied through the at least one cleaningfluid supply line (they may have separate or common supply lines) intothe cleaning fluid container. Cleaning fluid may be added to the inletend of the intake channel in all the methods and sequences describedabove, or any other suitable method and sequence. Upon completion, thecleaning fluid may be drained out of the at least one drain line (thedrain can go to an onboard waste container, an external waste storagecontainer, or an external waste drain). In certain embodiments, a valvecontrols when the drain is open. In certain embodiments, the drain linecomprises a pump for actively moving waste from the cleaning fluidcontainer to its final destination.

In certain embodiment, the at least one cleaning fluid line ispositioned so that a jet of fluid exits at least one cleaning fluid lineand impinges on the intake channel so that it may wash or substantiallywash any contaminating material, such as detectable or potentiallydetectable components, off of the intake channel and into the cleaningfluid reservoir. In certain embodiments, the at least one cleaning fluidline is positioned so that the outlet of the fluid line is submerged inthe cleaning fluid in the cleaning fluid container, and driving at leastone of the cleaning fluids through the cleaning fluid line inducesvorticity in the fluid in the cleaning fluid container that aids inwashing contaminating material, such as detectable or potentiallydetectable material off of the intake channel.

In certain embodiments, cleaning fluid is provided to the system throughthe intake channel by employing reverse flow. In these embodiments, theintake channel comprises, e.g., an inlet end, an injector, a pump, aselector valve, and a supply of at least one cleaning fluid. Afterinjection of an aliquot of sample fluid in the injector andre-positioning the injector so that the injector chamber (commonconduit) that contained the aliquot of sample fluid is once againrealigned with the intake channel, the selector valve is positioned sothat the supply of at least one cleaning fluid, the pump, the injector,and the inlet end of the intake channel are all aligned. A force isgenerated by the pump to drive flow of cleaning fluid from the cleaningfluid container, through the injector, and out the inlet end of theintake channel. In certain embodiments, at least one cleaning fluid hasa higher affinity for a surface material of the intake channel than thesample fluid, so the cleaning fluid displaces the sample fluid from theintake channel and drives all or substantially all of the sample fluidout of the inlet end of the intake channel. The cleaning fluid may bedriven into any of the cleaning fluid containers described above.

FIGS. 5A and 5B—System for cleaning an intake conduit FIG. 5 shows asystem for cleaning an intake conduit. The system comprises at least onecleaning fluid container 501, an aspiration conduit 502 (also referredto as an “intake conduit,” “intake line,” and similar wording, herein),and a sampler assembly 503. The cleaning fluid container comprises atleast one cleaning fluid. In certain embodiments, at least one of thecleaning fluids has a higher affinity for the surface of the aspirationconduit 502 than do dispersed phases in the system, allowing it todisplace dispersed phases within in the aspiration conduit 502. Incertain embodiments, the at least one cleaning fluid comprises an oiland the surface of the aspiration conduit 502 comprises a hydrophobicmaterial. In certain embodiments, the at least one cleaning fluidcomprises water and the surface of the aspiration conduit 502 comprisesa hydrophilic material. In certain embodiments, the cleaning fluidcomprises a fluorinated oil and the surface of aspiration conduit 502comprises a fluorinated polymer. In certain embodiments, at least one ofthe cleaning fluids comprises a denaturing fluid, such as a denaturingfluid comprising water and a component capable of rendering a detectableor potentially detectable component undetectable. In certainembodiments, the detectable component is a nucleic acid and thedenaturing component comprises any suitable denaturing fluid asdescribed herein for nucleic acids. In certain embodiments, at least oneof the cleaning fluids comprises a dilution fluid to reduce theconcentration of detectable or potentially detectable component. A“dilution fluid” is a purge fluid that is miscible with at least onedispersed phase fluid. In certain embodiments, the dilution fluidcomprises water. In certain embodiments, the dilution fluid comprises anoil. In certain embodiments, the cleaning fluid comprises a separation(spacer) fluid that is immiscible with at least one continuous phase andat least one dispersed phase. The separation fluid separates volumes ofat least one dispersed phase fluid from each other when dispersed in theat least one continuous phase fluid. In certain embodiments, theaspiration conduit 502 comprises a tube. In certain embodiments, theaspiration conduit comprises a polymer tube, such as a fluoropolymertube. The cleaning fluid container may be any suitable container. Incertain embodiments, the at least one cleaning fluid container 501comprises a well of a microtiter plate, a test tube, a well, a cuvette,or a tray.

The sampler assembly positions the aspiration conduit 502 into thecleaning fluid container 501. In certain embodiments, this positioningcomprises moving the aspiration conduit 502 down or up into the cleaningfluid container 501. In certain embodiments, this positioning comprisesmoving the cleaning fluid container 501 down or up into the samplecontainer. In some embodiments, this positioning comprises moving theaspiration conduit 502 in a plane normal or substantially normal to thecentral axis of the aspiration conduit 502 so as to position it to allowit to enter the cleaning fluid container 501. In some embodiments, thispositioning comprises moving the cleaning fluid container 501 in a planenormal or substantially normal to the central axis of the aspirationconduit so as it position the cleaning fluid container 501 to allow theaspiration conduit 502 to enter the cleaning fluid container.

The system additionally comprises an injection conduit 504, aninjector/valve assembly 505, a waste conduit 506, a waste 507, a motiveforce source 508, and an analysis conduit 509. The injection conduit 504is in fluid communication with the aspiration conduit 502 and with theinjector/valve assembly 505. The injector/valve assembly comprises aconduit that may exist in at least two states. In a first state, theconduit is in fluid communication with the injection conduit 504 and thewaste conduit 506. In a second state, the conduit is in fluidcommunication with the analysis conduit 509. The conduit is in at mostone of these states at any time.

In certain embodiments, such as shown in FIG. 5A, a method for cleaningthe system to avoid cross-contamination between volumes of at least onedispersed phase comprises positioning the aspiration conduit 502 in thecleaning fluid container 501 such that the inlet of the aspirationconduit 502 is submerged in the cleaning fluid. The injector/valveassembly conduit is positioned such that the injection conduit 504 andthe waste conduit 506 are in fluid communication. The motive forcesource 508 is actuated so as to create a suction to draw cleaningsolution into the aspiration conduit 502 through the injector conduit504 to the waste conduit 506 and into the waste container 507. Incertain embodiments, at least one cleaning fluid has a higher affinityfor a material comprising the internal surfaces of the aspirationconduit 502, the injector conduit 504, and the injector/valve assembly505 than for any dispersed phase in the system, allowing the at leastone cleaning fluid to displace dispersed phase from the surfaces of theconduits and into the waste container 507. In some embodiments, aplurality of cleaning solutions and/or cleaning solution containers isused, where the final cleaning solution aspirated in the method has ahigher affinity for a material comprising the internal surfaces of theaspiration conduit 502, the injector conduit 504, and the injector/valveassembly 505 so as to displace dispersed phase from the surfaces of theconduits and into the waste container 507 and avoid cross contamination.The motive force source 508 may be positioned at any point in the systemso as to create a suction in the aspiration conduit 502. In someembodiments, the motive force source 508 is positioned between theinjector/valve assembly 505 and the waste container 507 so that thewetted surfaces of the motive force source 508 do not contact fluid thatwould enter the analysis conduit 509 when the injector/valve ispositioned so as to place its conduit in fluid communication with theanalysis conduit 509. In some embodiments, the motive force source 508is a pump. In further embodiments, the pump is a peristaltic pump, adiaphragm pump, a centrifugal pump, a syringe pump, a positivedisplacement pump, or a reciprocating pump.

In certain embodiments, such as shown in FIG. 5B, the system comprises apurge/clean conduit 510 and at least one purge/clean fluid reservoir511. The injector/valve assembly 505 comprises a conduit that may existin at least two states. In a first state, the conduit is in fluidcommunication with the injection conduit 504 and the purge/clean conduit510. In a second state, the conduit is in fluid communication with theanalysis conduit 509. The conduit may be in at most one state at anytime.

A method for cleaning the system so that volumes of at least onedispersed phase are not cross-contaminated comprises positioning theaspiration conduit 502 with the autosampler assembly 503 so that fluidleaving the aspiration conduit will deposit in the cleaning fluidcontainer 507, positioning the injector/valve assembly conduit such thatthe injection conduit 504 is in fluid communication with the purge/cleanconduit 510, actuating the motive force source 508 so as to drive fluidfrom the purge/clean reservoir 511 through the purge/clean conduit 510,the injector/valve assembly conduit 504, the aspiration conduit 502, andinto the cleaning fluid container 507. In some embodiments, at least onecleaning fluid has a higher affinity for a material comprising theinternal surfaces of the aspiration conduit 502, the injector conduit504, the injector/valve assembly 505, and the purge/clean conduit 510than for any dispersed phase, allowing the at least one cleaning fluidto displace dispersed phase from the surfaces of the conduits and intothe at least one cleaning fluid container 507. In certain embodiments, aplurality of cleaning solutions and/or cleaning solution containers isused, where the final cleaning solution aspirated in the method has ahigher affinity for a material comprising the internal surfaces of theaspiration conduit 502, the injector conduit 504, the injector/valveassembly 505, and the purge/clean conduit 510 so as to displacedispersed phase from the surfaces of the conduits and into the cleaningfluid container 507 and avoid cross contamination between volumes of theat least one dispersed phase.

In certain embodiments, the cleaning fluid container 507 comprises adrain such that cleaning fluids deposited in the cleaning fluidcontainer 507 through the aspiration conduit 502 may periodically orcontinuously be removed through an outlet to a waste. In otherembodiments, the at least one cleaning fluid container 507 does notcomprise a drain, and the system comprises an aspiration device forperiodically or continuously removing cleaning fluids deposited in theat least one cleaning fluid container 507 through the aspiration conduit502. In other embodiments, the cleaning fluid container 507 does notcomprise a drain, and the at least one cleaning fluid container 507 isdisposable so that cleaning fluids deposited in the at least onecleaning fluid container 507 may be disposed of by disposing of the atleast one cleaning fluid container 507.

FIGS. 6A and 6B shows further embodiments for both intaking a firstvolume of a dispersed phase and subsequently cleaning the intake systemso that other volumes of dispersed phase are not cross-contaminated bythe first volume of dispersed phase. The system additionally comprises adispersed phase container 614, and the autosampler assembly 603 iscapable of causing components comprising the system to be positionedsuch that the aspiration tip 602 may aspirate fluids from the dispersedphase container 614 or aspirate fluids from or dispense fluids into thecleaning fluid container 601. The dispersed phase container may containat least one dispersed phase. In certain embodiments, the dispersedphase comprises a biological or chemical sample, a biological orchemical reaction mixture, a biological or chemical assay, or a chemicalor biochemical reagent. In certain embodiments, the dispersed phasecomprises a nucleic acid, PCR reagents, reporter molecules, a protein,an antibody, a salt, glycerol, a surfactant, or combinations thereof. Amethod using the system of FIG. 6A comprises positioning componentscomprising the system such that the aspiration tip 602 may aspirate avolume of a dispersed phase in the dispersed phase container 614,positioning the injector/valve assembly 605 conduit such that theinjection conduit 604 is in fluid communication with the waste conduit606, actuating the motive force source 608 so that a first volume ofdispersed phase fluid is aspirated and at least partially fills thevolume of the injector/valve assembly 605 conduit, positioning theinjector/valve assembly 605 conduit so that it is in fluid communicationwith the analysis conduit 609, actuating the analysis fluid source 615so that a substantial first fraction of the dispersed phase fluid in theinjector/valve assembly 605 conduit is displaced into the analysisconduit, positioning the injector/valve 605 conduit such that it is influid communication with the injector conduit 604 and the waste conduit606, positioning components comprising the system such that theaspiration conduit 602 may aspirate a volume of at least one cleaningfluid from the at least one cleaning fluid reservoir 601, actuating themotive force source 608 to aspirate a volume of at least one cleaningfluid such that the at least one cleaning fluid passes through theaspiration conduit 602, the injection conduit 604, the waste conduit606, and into the waste reservoir 607, such that a substantial secondfraction of any volume, e.g., any residual volume, of at least onedispersed phase is displaced from the aspiration conduit 602, theinjection conduit 604, and the injector/valve assembly 605 conduit andinto the waste conduit 606 or the waste reservoir 607 andcross-contamination of subsequent volumes of dispersed phase fluid arecross-contaminated by the first volume of dispersed phase fluid. Incertain embodiments, the first fraction is greater than 70%, greaterthan 80%, greater than 90%, greater than 95%, greater than 99%, greaterthan 99.9%, greater than 99.99%, greater than 99.999%, or greater than99.9999%. In certain embodiments, the second fraction is greater than70%, greater than 80%, greater than 90%, greater than 95%, greater than99%, greater than 99.9%, greater than 99.99%, greater than 99.999%, orgreater than 99.9999%. In certain embodiments, a plurality of cleaningfluids may be aspirated from a plurality of cleaning fluid containers.In certain embodiments, a first cleaning fluid is aspirated from a firstcleaning fluid container 601 and a second cleaning fluid is aspiratedfrom a second cleaning fluid container. This may be extended to three,four, or any other suitable number of more pairs of cleaning fluids andcleaning fluid reservoirs. In certain embodiments, more than onecleaning fluid is aspirated from a single cleaning fluid container. In afurther embodiment, a first cleaning fluid has a gravimetric densitythat differs from at least one other cleaning fluid, and the cleaningfluids are contained in the same cleaning fluid container. Aspiration ofdifferent cleaning fluids is achieved by adjusting components comprisingthe system such that the tip of the aspiration conduit 602 may aspiratefluid of the respective cleaning fluid, aspirating fluid so that theinterface between two cleaning fluids drops such that the tip of theaspiration conduit 602 shifts from aspirating a first cleaning fluid toa second cleaning fluid, or some combination thereof.

In certain embodiments, the injector/valve assembly 605 comprises afirst conduit and a second conduit. The first conduit may exist in atleast two states. In the first state, the first conduit is positionedsuch that the injection conduit 604 is in fluid communication with thewaste conduit 606. In the second state, the first conduit is positionedsuch that the first conduit is in fluid communication with the analysisconduit 609. The second conduit may exist in at least two states. In thefirst state, the second conduit is positioned such that the injectionconduit 604 is in fluid communication with the waste conduit 606. In thesecond state, the second conduit is positioned such that the secondconduit is in fluid communication with the analysis conduit 609. Thefirst and second conduit may each exist in at most one of theirrespective first and second states at any time. Additionally, if thefirst conduit is in the first state, the second conduit is not in thefirst state; if the first conduit is in the second state, the secondconduit is not in the second state; if the second conduit is in thefirst state, the first conduit is not in the first state; if the secondconduit is in the second state, the first conduit is not in the secondstate. The first conduit may be in the first state while the secondconduit is in the second state; the first conduit may be in the secondstate while the second conduit is in the first state; the second conduitmay be in the first state while the first conduit is in the secondstate; and the second conduit may be in the second state while the firstconduit is in the first state. In some embodiments, the conduits may notbe in either the first state or the second state for a finite time. Infurther embodiments, the conduits are not in any state for a finite timewhile transitioning between states.

A method using the system of FIG. 6A where the injector/valve assembly605 comprises at least two conduits where, when the first conduit is inthe first state, the second conduit is in the second state, and when thesecond conduit is in the first state, the first conduit is in the secondstate, comprises positioning components of the system such that theaspiration tip 602 may aspirate a volume of a dispersed phase in thedispersed phase container 614, positioning the injector/valve assembly605 first conduit such that the injection conduit 604 is in fluidcommunication with the waste conduit 606, actuating the motive forcesource 608 so that a first volume of dispersed phase fluid is aspiratedand at least partially fills the volume of the injector/valve assembly605 first conduit and positioning the injector/valve assembly 605 firstconduit so that it is in fluid communication with the analysis conduit609. The method further comprises actuating the analysis fluid source615 so that a substantial first fraction of the dispersed phase fluid inthe injector/valve assembly 605 first conduit is displaced into theanalysis conduit (process side conduit) and simultaneously positioningcomponents comprising the system such that the aspiration conduit (tip)602 may aspirate a volume of at least one cleaning fluid from the atleast one cleaning fluid reservoir 601, actuating the motive forcesource 608 to aspirate a volume of at least one cleaning fluid such thatthe at least one cleaning fluid passes through the aspiration conduit602, the injection conduit 604, the waste conduit 606, and into thewaste reservoir 607, such that a substantial second fraction of anyvolume, e.g., any residual volume, of at least one dispersed phase isdisplaced from the aspiration conduit 602, the injection conduit 604,and the injector/valve assembly 605 second conduit and into the wasteconduit 606 or the waste reservoir 607 and cross-contamination ofsubsequent volumes of dispersed phase fluid are cross-contaminated bythe first volume of dispersed phase fluid. The method further comprisespositioning the first conduit of the injector/valve assembly 605 suchthat the injection conduit 605 and the waste conduit 606 are in fluidcommunication, positioning components of the system such that theaspiration conduit 602 is may aspirate at least one cleaning fluid fromat least one cleaning fluid reservoir, actuating the motive force source608 such that at least one volume of at least one cleaning fluid isaspirated into the aspiration conduit 602, through the injection conduit604 and injector/valve assembly 605 first conduit and into the wasteconduit 606 or waste reservoir 607 such that a substantial thirdfraction of any remaining volume of dispersed phase is displaced fromthe aspiration conduit 602, injection conduit 604 and injector/valveassembly 605 first conduit, reducing the potential forcross-contamination between the first volume of dispersed phase andother volumes of dispersed phase. In certain embodiments, the firstfraction is greater than 70%, greater than 80%, greater than 90%,greater than 95%, greater than 99%, greater than 99.9%, greater than99.99%, greater than 99.999%, or greater than 99.9999%. In certainembodiments, the second fraction is greater than 70%, greater than 80%,greater than 90%, greater than 95%, greater than 99%, greater than99.9%, greater than 99.99%, greater than 99.999%, or greater than99.9999%. In certain embodiments, the third fraction is greater than70%, greater than 80%, greater than 90%, greater than 95%, greater than99%, greater than 99.9%, greater than 99.99%, greater than 99.999%, orgreater than 99.9999%. In certain embodiments, a plurality of cleaningfluids may be aspirated from a plurality of cleaning fluid containers.In certain embodiments, a first cleaning fluid is aspirated from a firstcleaning fluid container 601 and a second cleaning fluid is aspiratedfrom a second cleaning fluid container. This may be extended to three,four, or any suitable number of more pairs of cleaning fluids andcleaning fluid reservoirs. In other embodiments, more than one cleaningfluid is aspirated from a single cleaning fluid container. In a furtherembodiment, a first cleaning fluid has a gravimetric density thatdiffers from at least one other cleaning fluid, and the cleaning fluidsare contained in the same cleaning fluid container. Aspiration ofdifferent cleaning fluids is achieved by adjusting components comprisingthe system such that the tip of the aspiration conduit 602 may aspiratefluid of the respective cleaning fluid, aspirating fluid so that theinterface between two cleaning fluids drops such that the tip of theaspiration conduit 602 shifts from aspirating a first cleaning fluid toa second cleaning fluid, or some combination thereof.

In embodiments of certain methods, air may be aspirated before or aftervolumes of dispersed phase or cleaning fluids. This may be helpful whenthe volume of the aspiration conduit 602, the injection conduit 604, andone of the injector/valve assembly 605 conduits exceeds the volume ofdispersed phase, at least one cleaning fluid, or any combinationthereof, available to be aspirated. In some further embodiments, thesystem comprises a sensor to detect the boundaries between air andvolumes of dispersed phase or at least one cleaning fluid or both.

A method using the system of FIG. 6B comprises positioning components ofthe system such that the aspiration tip 602 may aspirate a volume of adispersed phase in the dispersed phase container 614, positioning theinjector/valve assembly 605 conduit such that the injection conduit 604is in fluid communication with the purge/clean conduit 610, actuatingthe motive force source 608 so that a first volume of dispersed phasefluid is aspirated and at least partially fills the volume of theinjector/valve assembly 605 conduit, positioning the injector/valveassembly 605 conduit so that it is in fluid communication with theanalysis conduit 609, actuating the analysis fluid source 615 so that afirst fraction of the dispersed phase fluid in the injector/valveassembly 605 conduit is displaced into the analysis conduit, positioningthe injector/valve 605 conduit such that it is in fluid communicationwith the injector conduit 604 and the waste conduit 606, positioningcomponents comprising the system such that the aspiration conduit 602may dispense a volume of at least one cleaning fluid into the least onecleaning fluid container 611, actuating the motive force source 608 toflow a volume of at least one cleaning fluid from the purge/cleanreservoir such that the at least one cleaning fluid passes through thepurge/clean conduit 610, the injection conduit 604, the aspirationconduit 602, and into the cleaning fluid container 607, such that asecond fraction of any volume of at least one dispersed phase isdisplaced from the aspiration conduit 602, the injection conduit 604,and the injector/valve assembly 605 conduit and into the cleaning fluidcontainer 607 and subsequent volumes of dispersed phase fluid are notcross-contaminated by the first volume of dispersed phase fluid. Incertain embodiments, the first fraction is greater than 70%, greaterthan 80%, greater than 90%, greater than 95%, greater than 99%, greaterthan 99.9%, greater than 99.99%, greater than 99.999%, or greater than99.9999%. In certain embodiments, the second fraction is greater than70%, greater than 80%, greater than 90%, greater than 95%, greater than99%, greater than 99.9%, greater than 99.99%, greater than 99.999%, orgreater than 99.9999%. In certain embodiments, a plurality of cleaningfluids may be flowed from a plurality of purge/clean reservoirs. In someembodiments, a first cleaning fluid may be flowed from a firstpurge/clean reservoir, and a second cleaning fluid may subsequently beflowed from a second purge/clean reservoir. This may extend to anysuitable additional number of purge/clean reservoirs.

A method using the system of FIG. 6B where the injector/valve assembly605 comprises at least two conduits where, when the first conduit is inthe first state, the second conduit is in the second state, and when thesecond conduit is in the first state, the first conduit is in the secondstate, comprises positioning components comprising the system such thatthe aspiration tip 602 may aspirate a volume of a dispersed phase in thedispersed phase container 614, positioning the injector/valve assembly605 first conduit such that the injection conduit 604 is in fluidcommunication with the purge/clean conduit 610, actuating the motiveforce source 608 so that a first volume of dispersed phase fluid isaspirated and at least partially fills the volume of the injector/valveassembly 605 first conduit and positioning the injector/valve assembly605 first conduit so that it is in fluid communication with the analysisconduit 609. The method further comprises actuating the analysis fluidsource 615 so that a first fraction of the dispersed phase fluid in theinjector/valve assembly 605 first conduit is displaced into the analysisconduit and simultaneously positioning components of the system suchthat the aspiration conduit 602 may dispense a volume of at least onecleaning fluid into the at least one cleaning fluid reservoir 601,actuating the motive force source 608 to flow a volume of at least onecleaning fluid such that the at least one cleaning fluid passes throughthe purge/clean conduit 610, the injection conduit 604, the aspirationconduit 602, and into the cleaning fluid container 601, such that asecond fraction of any volume of at least one dispersed phase isdisplaced from the aspiration conduit 602, the injection conduit 604,and the injector/valve assembly 605 second conduit and into the cleaningfluid container 601 and cross-contamination of subsequent volumes ofdispersed phase fluid by the first volume of dispersed phase fluid isreduced or eliminated. The method further comprises positioning thefirst conduit of the injector/valve assembly 605 such that the injectionconduit 604 and the purge/clean conduit 610 are in fluid communication,positioning components comprising the system such that the aspirationconduit 602 may dispense at least one cleaning fluid into at least onecleaning fluid reservoir, actuating the motive force source 608 suchthat at least one volume of at least one cleaning fluid is flowed intothe purge/clean conduit 606, through the injection conduit 604 andinjector/valve assembly 605 first conduit and into the cleaning fluidreservoir 601 such that a third fraction of any remaining volume ofdispersed phase is displaced from the aspiration conduit 602, injectionconduit 604 and injector/valve assembly 605 first conduit, reducing thepotential for cross-contamination between the first volume of dispersedphase and other volumes of dispersed phase. In certain embodiments, thefirst fraction is greater than 70%, greater than 80%, greater than 90%,greater than 95%, greater than 99%, greater than 99.9%, greater than99.99%, greater than 99.999%, or greater than 99.9999%. In someembodiments, the second fraction is greater than 70%, greater than 80%,greater than 90%, greater than 95%, greater than 99%, greater than99.9%, greater than 99.99%, greater than 99.999%, or greater than99.9999%. In some embodiments, the third fraction is greater than 70%,greater than 80%, greater than 90%, greater than 95%, greater than 99%,greater than 99.9%, greater than 99.99%, greater than 99.999%, orgreater than 99.9999%. In some embodiments, a plurality of cleaningfluids may be aspirated from a plurality of cleaning fluid containers.In some embodiments, a first cleaning fluid is aspirated from a firstcleaning fluid container 601 and a second cleaning fluid is aspiratedfrom a second cleaning fluid container. This may be extended to three,four, or arbitrarily more pairs of cleaning fluids and cleaning fluidreservoirs.

FIG. 7—System for injecting a sample comprising a waste station FIG. 7shows a further embodiment of the systems shown in FIG. 6B. The systemfurther comprises a washing conduit 712 and the autosampler assembly 703comprises a source of washing fluid in fluid communication with thewashing conduit 712 and a second motive force source to drive thewashing fluid through the washing conduit 712. The washing conduit 712is positioned so that the inner surface of the washing conduit 712substantially surrounds at least part of the outer surface of theaspiration conduit 701 but terminates at a vertical position above a tipof the aspiration conduit 701. An embodiment of a method of preventingcross-contamination using the system of FIG. 7 comprises positioning theaspiration conduit 702 with the autosampler assembly 703 such thatfluids leaving the aspiration conduit and the washing conduit 712deposit in the at least one cleaning fluid container 701, positioningthe injector/valve assembly 705 conduit such that the injection conduit704 is in fluid communication with the purge/clean conduit 706,actuating the motive force source 708 to move at least one cleaningfluid from the purge/clean reservoir 709 through the purge clean conduit708, the injection conduit 704, and the aspiration conduit 702 so thatthe cleaning fluids are deposited in the at least one cleaning fluidcontainer 701. The second motive force source is actuated to drive thewashing fluid through the washing fluid conduit 712 and displace volumesof the at least one dispersed phase from the outer surface of theaspiration conduit 702 and into the cleaning fluid container 701. Insome embodiments, the washing fluid has a higher affinity for the outersurface of the aspiration conduit 702 than the at least one dispersedphase so that the washing fluid may preferentially drive the at leastone dispersed phase from the outer surface of the aspiration conduit702. In some embodiments, the washing fluid comprises a component thatis hydrophobic, the outer surface of the aspiration conduit 702comprises a component that is hydrophobic, and the at least onedispersed phase comprises water. In an embodiment, the washing fluid isan oil and the outer surface of the aspiration conduit 702 comprises apolymer. In a preferred embodiment, the washing fluid is a fluorinatedoil and the outer surface of the aspiration tube comprises a fluorinatedpolymer. In other embodiments, the washing fluid comprises a componentthat is hydrophilic, the outer surface of the aspiration tube 702comprises a component that is hydrophilic, and the at least onedispersed phase comprises a component that is hydrophobic.

In some embodiments, the method additionally comprises flowing washingthrough the washing fluid conduit 712 and into the cleaning fluidcontainer 701, re-positioning the aspiration tube 702 such that a tip ofthe aspiration tube 702 is vertically above the surface of at least onecleaning fluid in the cleaning fluid container 701, and then terminatingflow of the washing fluid conduit 712 into the cleaning fluid container701 such that the washing fluid continues to flow until the tip of theaspiration tube 702 is no longer in fluid communication with at leastone cleaning fluid.

A method of sampling and cleaning an intake system at least onedispersed phase from at least one dispersed phase container using thesystem of FIG. 7 comprises positioning components comprising the systemsuch that the aspiration tip 702 may aspirate a volume of a dispersedphase in the dispersed phase container 713, positioning theinjector/valve assembly 705 conduit such that the injection conduit 704is in fluid communication with the purge/clean conduit 710, actuatingthe motive force source 708 so that a first volume of dispersed phasefluid is aspirated and at least partially fills the volume of theinjector/valve assembly 705 conduit, positioning the injector/valveassembly 705 conduit so that it is in fluid communication with theanalysis conduit 707, actuating the analysis fluid source 715 so that asubstantial first fraction of the dispersed phase fluid in theinjector/valve assembly 705 conduit is displaced into the analysisconduit, positioning the injector/valve 705 conduit such that it is influid communication with the injector conduit 704 and the waste conduit706, positioning components comprising the system such that theaspiration conduit 702 may dispense a volume of at least one cleaningfluid into the least one cleaning fluid container 701, actuating themotive force source 708 to flow a volume of at least one cleaning fluidfrom the purge/clean reservoir such that the at least one cleaning fluidpasses through the purge/clean conduit 710, the injection conduit 704,the aspiration conduit 706, and into the cleaning fluid container 701,such that a substantial second fraction of any volume of at least onedispersed phase is displaced from the aspiration conduit 702, theinjection conduit 704, and the injector/valve assembly 705 conduit andinto the cleaning fluid container 701, flowing a washing fluid throughthe washing fluid 712 and into the cleaning fluid container 701 suchthat the washing fluid displaces dispersed phase fluid on the outersurface of the aspiration conduit 702 and into the cleaning fluidcontainer 701 and cross-contamination of subsequent volumes of dispersedphase fluid are cross-contaminated by the first volume of dispersedphase fluid. In some embodiments, the first fraction is greater than70%, greater than 80%, greater than 90%, greater than 95%, greater than99%, greater than 99.9%, greater than 99.99%, greater than 99.999%, orgreater than 99.9999%. In some embodiments, the second fraction isgreater than 70%, greater than 80%, greater than 90%, greater than 95%,greater than 99%, greater than 99.9%, greater than 99.99%, greater than99.999%, or greater than 99.9999%. In some embodiments, a plurality ofcleaning fluids may be flowed from a plurality of purge/cleanreservoirs. In some embodiments, a first cleaning fluid may be flowedfrom a first purge/clean reservoir, and a second cleaning fluid maysubsequently be flowed from a second purge/clean reservoirs. This mayarbitrarily extend to any number of purge/clean reservoirs. Methods thatuse an injector/valve assembly 705 comprising at least two conduits anda system additionally comprising the washing conduit 712 are analogousto methods not additionally comprising the washing conduit 712,additionally comprising a step flowing washing fluid through the washingconduit 712 and into the cleaning fluid container 701 such that thewashing fluid displaces dispersed phase fluid from the outer surface ofthe aspiration tube 702.

FIG. 8A—Patterns for sampling to avoid cross-contamination FIGS. 8A and8B show a system for containing dispersed phase volumes (e.g., samples)and methods for avoiding carryover and cross-contamination whenaspirating consecutive volumes of dispersed phase. The system comprisesa set of dispersed phase containers 801 comprising at least twodispersed phase containers which may contain at least one dispersedphase. When aspirating dispersed phase volumes, a tip of an aspirationconduit is submerged in at least one dispersed phase volume in a firstdispersed phase container. The tip of the aspiration conduit issubsequently submerged in a second volume of dispersed phase in a seconddispersed phase container. In certain embodiments, the tip of theaspiration conduit may be cleaned in between submerging the tip of theaspiration conduit in the first dispersed phase volume and submergingthe tip of the aspiration conduit in the second dispersed phase volume,where the cleaning occurs at a site remote from the first and seconddispersed phase containers. A method for avoiding carryover of a firstdispersed phase fluid from a first dispersed phase fluid container to asecond dispersed phase fluid in a second dispersed phase fluid containerwhere an aspiration tip is cleaned at a location remote from both thefirst dispersed phase fluid container and the second dispersed phasefluid container comprises aspirating the first dispersed phase from thefirst dispersed phase container, moving to the remote location for tipcleaning where the movement does not traverse any position where theaspiration conduit tip is vertically positioned above the seconddispersed phase container, cleaning the aspiration conduit tip in theremote location, and positioning the aspiration conduit tip so that itis vertically positioned above the second dispersed phase container suchthat, in positioning, the aspiration conduit tip only traversespositions where the tip of the aspiration conduit is above a dispersedphase container if the aspiration tip has already been submerged in adispersed phase in that dispersed phase container. In certainembodiments, the dispersed phase containers are wells of a microtiterplate and the set 801 is a microtiter plate. Any suitable microtiterplate may be used; in certain embodiments, the microtiter plate is a24-well plate, a 48-well plate, a 96-well plate, a 384-well plate, or a1536-well plate. FIGS. 8A and B show sequential orders of aspiratingwells in a microtiter plate such that, when positioning to aspirate avolume of dispersed phase from a dispersed phase container, the tip ofthe aspiration conduit only ever traverses positions where it isvertically above another dispersed phase container if it has alreadybeen submerged in dispersed phase fluid in the dispersed phasecontainer.

FIGS. 9A and 9B—Examples of aspirating a fluid FIGS. 9A and 9B showexamples of aspirating a fluid from a system comprising a fluidcontainer 901 and an aspiration conduit 902 comprising a tip. Theinternal volume of the fluid container 901 comprises at least one firstfluid. When the tip is submerged in the at least one first fluid, boththe inner and outer surfaces of the aspiration conduit 902 are exposedto the at least one fluid. After aspiration of some or all of the fluid,a portion of the first fluid may remain on the outer surface of theaspiration conduit 902. The system comprises a second fluid container903, and subsequent aspiration of the fluid in the second fluid withoutwashing the outer surface of the aspiration conduit 902 (as in FIG. 9A)may result in cross-contamination of the second fluid by the firstfluid. Instead, if the outer surface of the aspiration conduit 902 iswashed (as in FIG. 9B) such that a portion of any of the first fluidremaining on the outer surface of the aspiration conduit 902 is removed,and the potential for cross-contamination of the second fluid by the atleast one first fluid is reduced. In some embodiments, the portion offirst fluid that is removed is greater than 70%, greater than 80%,greater than 90%, greater than 95%, greater than 98%, greater than 99%,greater than 99.9%, greater than 99.99%, or greater than 99.999% of theat least one first fluid originally on the outer surface of theaspiration conduit 902. In certain embodiments, at least one washingfluid is used to wash the aspiration conduit, where the washing fluidhas a higher affinity for a material comprising the outer surface of theaspiration conduit 902 than does the at least one first fluid. Incertain embodiments, the outer surface of the aspiration conduitcomprises a hydrophobic polymer, the at least one first fluid compriseswater, and the washing fluid comprises a hydrophobic component. Incertain embodiments, the surface of the aspiration conduit comprises afluoropolymer, the at least one first fluid comprises water, and thewashing fluid comprises a fluorinated oil.

FIGS. 10A and 10B—Examples of injecting a sample from a sample containerwith a cover FIGS. 10A and 10B show systems for aspirating fluids fromsample containers where a cover prevents carryover of one sample intoanother. The system comprises a first fluid container whose volumecomprises at least one first fluid; a cover 1003; and an aspirationconduit 1002 where the aspiration conduit 1002 may pass through thecover in such a way that a substantial portion of the at least one firstfluid is removed from the outer surface of the aspiration conduit 1002as it passes outside of the first fluid container. In certainembodiments, the cover 1003 is a polymer seal. In certain embodiments,the cover 1003 is a silicone seal, and the aspiration conduit 1002 iscapable of pushing through the silicone seal, where the silicone sealwipes the outer surface of the aspiration conduit 1002 to remove thefirst fluid. In some embodiments, the portion of the at least one firstfluid removed from the aspiration conduit is greater than 70%, greaterthan 80%, greater than 90%, greater than 95%, greater than 98%, greaterthan 99%, greater than 99.9%, greater than 99.99%, or greater than99.999% of the first fluid that remains on the aspiration conduit afterit samples the first fluid.

FIGS. 4A, 4B, 4C, and 4D—Layered fluids in sample containers FIG. 4shows multiple systems for providing dispersed phase fluids and/orcleaning fluids to the system, where the fluids share a common containerand are separated by, e.g., differences in mass density (e.g., parfait).The system comprises a fluid container 401 and at least one fluid 402.In FIG. 4A, the system only comprises a first fluid 402. In FIG. 4B, thesystem additionally comprises a second fluid 403 that is different fromthe first fluid and that has a lower mass density than the first fluid402. The first and second fluids may independently be, e.g., continuousor dispersed phase fluids in the system. FIG. 4C shows where the systemcomprises two fluids, but that it comprises a third fluid 404 that has alower mass density than the fluid 402. FIG. 4D shows a system thatcomprises three different fluids: a first fluid 402, a second fluid 403,and a third fluid 404. By deliberate choice of the fluids, dispersedphase fluids and cleaning fluids may be sequentially added to thesystem.

In certain embodiments, the first fluid 402 is a first dispersed phasefluid that comprises, e.g., an analyte or other component to be passedto a process system for processing, the second fluid 403 is a seconddispersed phase fluid immiscible with the first dispersed phase, and thethird fluid 404 is a continuous phase fluid. Layering the second fluid403 on top of the first fluid 402 prevents the first fluid 402 fromevaporating until the second fluid 403 has substantially evaporated orhas been removed. Layering the third fluid 404 under the first fluid 402raises the vertical position of the first fluid 402, reducing thedifficulty of aspirating most or all of the first fluid 402 from thebottom of the fluid container 402. In certain embodiments, the secondfluid 403 is also a spacer fluid to separate distinct volumes of firstfluid aspirated into an aspiration conduit. In certain embodiments, thethird fluid 404 is a purge fluid to displace volumes of first fluid froman aspiration and intake system, reducing the possibility ofcross-contamination between distinct volumes of first fluid.

In certain embodiments, a single position of a tip of an aspirationconduit allows for aspirating a sequence of fluids. For example,positioning a tip of an aspiration conduit such that it is submerged inthe third fluid 404 and then beginning aspiration will first draw thirdfluid 404 into the aspiration conduit, vertically lowering the interfacebetween the third fluid 404 and the first fluid 402 until the aspirationtip is submerged in the first fluid 402. Further aspiration aspiratesfirst fluid 402 into the aspiration conduit, vertically lowering theinterface between the first fluid 402 and the second fluid 403 until theaspiration tip is submerged in the second fluid 403. Further aspirationaspirates second fluid into the aspiration conduit. In certainembodiments, systems and methods include sequentially positioning a tipof an aspiration conduit such that it is sequentially submerged in atleast two fluids allows for the sequential ordering of aspiration of theat least two fluids.

FIGS. 11A and 11B—Systems and methods for creating layered fluids insample containers FIG. 11 shows systems and methods for dispensingfluids to create the vertically layered fluids (parfait) shown in FIG.4. The system comprises a fluid container 1101, an aspiration conduit1102, a fluid source 1102, and a first fluid 1104. In a method shown inFIG. 11A, the aspiration conduit 1102 is positioned so that a tip of theaspiration conduit 1102 is submerged in the first fluid 1104. A secondfluid 1105 with a mass density greater than the mass density of thefirst fluid 1104 is provided by the fluid source 1103, flowed throughthe aspiration conduit 1102 through the tip of the aspiration conduit1102 and into the fluid container 1101, where it settles to the bottomof the fluid container 1101. A third fluid 1106 with a mass density lessthan the mass density of the first fluid is provided by the fluid source1103, flowed through the aspiration conduit 1102 and through the tip ofthe aspiration conduit 1102 and into the fluid container 1101, where itfloats to the top of the first fluid 1104.

In a method shown in FIG. 11B, the aspiration conduit 1102 is positionedso that a tip of the aspiration conduit 1102 is submerged in the firstfluid 1104. The third fluid 1106 is first provided by the fluid source1103, flowed through the aspiration conduit 1102 through the tip of theaspiration conduit 1102 and into the fluid container 1101, where itfloats to the top of the fluid container 1101. The second fluid 1105 isprovided by the fluid source 1103, flowed through the aspiration conduit1102 and through the tip of the aspiration conduit 1102 and into thefluid container 1101, where it settles to the bottom of the fluidcontainer 1101.

In certain embodiments, the rate of volumetric flow through theaspiration conduit 1102 of the second fluid 1105 and third fluid 1106 islimited so that well-defined interfaces are maintained between thefirst, second, and third fluids. In preferred embodiments, the rate ofvolumetric flow is less than 1000 mL/min, less than 100 mL/min, or lessthan 10 mL/min.

In certain embodiments, the fluid source comprises a first reservoir forthe second fluid 1105, a second reservoir for the third fluid 1106, atleast one fluid selection valve, and a motive force source. The at leastone fluid selection valve causes one or neither (but not both) of thesecond fluid 1105 or third fluid 1106 to flow when a motive force isprovided by the motive force source. In some embodiments, the motiveforce source is a pump.

FIG. 12—System for sensing the level of a fluid with a sampling inlet.In certain embodiments of the systems and methods provided herein, it isdesirable to determine when a tip of an aspiration conduit is submergedin a fluid volume. In certain embodiments, this is to ensure aspirationof sufficient volume, to avoid aspirating more than a maximum volume ofair, or to ensure a fluid container contains a minimum volume of fluidbefore aspirating. The system shown in FIG. 12 comprises an aspirationconduit 1201 and an aspiration tip 1202 that comprises a fluid sensingdevice 1203. A signal from the fluid sensing device 1204 indicateswhether the aspiration tip 1202 is submerged in a fluid. The fluidsensing device may be any suitable device; in certain embodiments, thefluid sensing device is an electrical resistance sensor, an electricalcapacitance sensor, a thermal conductivity sensor, a heat capacitysensor, a nuclear or particulate radiation sensor, or a temperaturesensor, or a combination thereof.

FIGS. 13A and 13B—Design of seal to avoid sample contamination Incertain embodiments of the systems and methods provided herein, fluidcontainers are sealed so as to avoid evaporation of fluid contents orenvironmental contamination or both. In order to aspirate the fluidcontents, the seal must be broken and an aspiration conduit insertedinto the fluid contents volume. In FIG. 13, a sealed fluid containersystem comprises a fluid container 1301, a seal 1302, and fluid contents1303. The system comprises a distance a from the inner surface of theseal 1302 to the top surface of the fluid contents and a maximum sealchord b. Because the outer surface of the seal 1302 is exposed to theambient environment, environmental contaminants may accumulate on theouter surface of the seal 1302 before the seal 1302 is broken and thefluid contents aspirated. If the distance a is less than or equal to amultiple of the distance b, breakage of the seal 1302 may result in aseal fragment 1304 being submerged in the fluid contents 1303,potentially contaminating the fluid contents 1303 with environmentalcontaminants from the outer surface of the seal 1302 (FIG. 13A). If thedistance a is greater than a multiple of the distance b, breakage of theseal 1302 will not result in a seal fragment 1304 being submerged in thefluid contents (FIG. 13B). At a minimum, the multiple must be 0.5. Insuch an embodiment, the seal must be perfectly broken such that no sealfragment 1304 has a longer segment than any other seal fragment. Inpreferred embodiments, the multiple is greater than 1, which guaranteesthat no seal fragment 1304 will be submerged in the fluid contents 1303.

FIGS. 14A and 14B—Sealing systems FIG. 14 shows systems for seals andbreaking seals. The system in FIG. 14 comprises a fluid container 1401,a seal 1402 where the seal has been perforated to require a reducedbreaking force and fluid contents 1403. Because the seal has beenperforated to require a reduced breaking force, an aspiration conduitrequires lower rigidity to break the seal than when there is noperforation (FIGS. 14A and 14B) In embodiments where the seal does notcomprise a perforation, the system may additionally comprise a sealbreaker distinct from an aspiration conduit to provide the force andrigidity required to break the seal.

FIGS. 15A, 15B, and 15C—Systems for aspirating samples and piercingseals FIG. 15 shows various systems for aspirating samples and piercingseals. The system in FIG. 15A comprises an aspiration conduit 1501 thatcomprises a tip 1502 and a piercing/aspiration assembly 1503. Theaspiration tip 1502 is sufficiently rigid to pierce a fluid containerseal used in the system, and the piercing aspiration assembly 1503allows for vertical actuation of the assembly and creation of suction atthe tip 1502 so as to aspirate fluids into the aspiration conduit 1501.The system in FIG. 15B comprises an aspiration conduit 1501 thatcomprises a tip 1502, where the tip 1502 is not sufficiently rigid ormechanically robust to pierce a fluid container seal used in the system.The system additionally comprises an aspiration assembly 1503 thatallows for vertical actuation of the tip 1502 and creation of suction atthe tip 1502 to aspirate fluids into the aspiration conduit 1501, apiercing tip 1504 and piercing assembly 1505, where the piercingassembly 1505 allows for vertical actuation of the piercing tip 1504 andcreation of the force required to pierce a seal in the system. In anembodiment of a method to employ the system in FIG. 15B, the piercingtip 1504 is positioned above a seal in the system and actuated downwardby the piercing assembly 1505 to break the seal. Once the seal is broke,the piercing assembly 1505 actuates the piercing tip 1504 upward, andthe tip 1502 is positioned above the fluid container. The tip 1502 ispositioned by the aspiration assembly 1503 downward into the fluidcontainer and fluid is aspirated by the aspiration assembly 1503 intothe aspiration conduit 1501. When aspiration is complete, the tip 1502is actuated upward by the aspiration assembly 1503.

In certain embodiments, the piercing tip 1504 has a round, star, square,conical, serrated, pyramidal, or rectangular cross section. In certainembodiments, the piercing tip 1504 comprises a metal, a polymer, or aglass, or a combination thereof. In certain embodiments, the surface ofthe piercing tip 1504 has a higher affinity for at least one continuousphase in the system than for any dispersed phase in the system. Incertain embodiments, the piercing tip comprises a fluoropolymer. Incertain embodiments, the tip comprises a fluoropolymer.

The system in FIG. 15C comprises an aspiration conduit 1501 comprising atip 1502, a piercing tip 1503 where the piercing tip substantiallysurrounds a portion of the aspiration conduit 1501, and anaspiration/piercing assembly 1504 that allows for independent verticalmotion of the tip 1502 and piercing tip 1503 as well as generation ofsuction at the tip 1502 to drive aspiration of fluids into theaspiration conduit 1501. A method for breaking a seal and aspirating afluid from a fluid container comprises positioning the tip 1502 above aseal of a fluid container, actuating the piercing tip 1503 downward sothat it pierces the seal but so that it does not become submerged influid contents of the fluid container, actuating the tip 1502 downwardso that it becomes submerged in fluid contents of the fluid container,and generating suction at the tip 1502 so as to aspirate part or all ofthe fluid contents into the aspiration conduit 1501. In certainembodiments, the piercing tip 1503 has a round cross-section, and thepiercing tip 1503 and the tip 1502 are co-axial or substantiallyco-axial.

FIG. 16—Design of an aspiration tip for filtration FIG. 16 shows anaspiration tip system for avoiding aspiration of particulate materialthat is oversized into the system. Particulate material with a largecharacteristic dimension may occlude fluid conduits in the system. Thesystem comprises an aspiration conduit 1501 with a tip 1502, such thatthe tip 1502 has a smaller cross-sectional area than other portions ofthe aspiration conduit 1501. The tip 1502 has a major dimension a. Toavoid aspiration of problematic particulate material, the majordimension a is chosen such that it is a fraction of the characteristicdimension of the smallest conduit in the present system. In certainembodiments, the major dimension is a diameter, a hydraulic diameter, orother suitable dimension. In certain embodiments, the majorcharacteristic dimension is a diameter, a hydraulic diameter, or othersuitable dimension. In some embodiments, a is less than 95%, less than90%, less than 85%, less than 80%, less than 70%, less than 50%, lessthan 35% or less than 20% of the characteristic dimension of thesmallest conduit in the present system. In an example, the smallestconduit is a rectangular channel with a minor axis that is 75 um, and ais a circular diameter chosen to be 50 um or less.

In certain embodiments, the aspiration conduit 1501 comprises a tubecomprising a polymer. In a certain embodiments, the tip is manufacturedby heating the aspiration conduit 1501 and applying a tensile force tothe aspiration conduit 1501 such that a portion of the length of theaspiration conduit 1501 has a reduced cross-sectional area andsubsequently cutting the aspiration conduit 1501 in the region ofreduced cross-sectional area so as to create the tip 1501. In certainembodiments, the tip is manufactured by inserting a mandrel of fixeddiameter into the internal volume of the aspiration conduit 1501,applying a compressive force to the aspiration conduit 1501 so as toreduce the cross-sectional area in a region of the aspiration conduit1501 where the cross-sectional area is the same as the mandrel in atleast part of the region, removing the mandrel, and cutting theaspiration conduit 1501 to create a tip 1502. In certain embodiments,the tip is manufactured by applying a compressive force to theaspiration conduit 1501 until the cross-sectional area of a portion ofthe aspiration conduit 1501 is zero, cutting the aspiration conduit 1501in the region of zero cross-sectional area, and creating a hole in theface of the aspiration conduit 150-1 where the aspiration conduit is cutto form a tip, where the hole provides fluidic communication between thetip 1502 and the balance of the internal volume of the aspirationconduit 1502. In certain embodiments, the holes are created by drilling,application of coherent electromagnetic radiation, or wire EDM. Incertain embodiments, the tip 1502 is manufactured by creating holes in asheet comprising a polymer material and welding the sheet to the end ofthe aspiration conduit 1501.

In certain embodiments, the tip 1502 comprises a hypodermic needle. Incertain embodiments, the surface of the needle comprises a material thathas a higher affinity for at least one continuous phase than anydispersed phase in the system.

FIGS. 17A and 17B—Methods for aspirating a fluid FIG. 17 shows methodsfor aspirating a sample in an intake system and transferring the sampleto a process system using an injector so that air is not injected intothe system. In an embodiment shown in FIG. 17A, a volume of sample drawninto the injector is checked to determine whether the volume of samplecomprises air. The detection method may measure an optical, ultrasonic,thermal, or other appropriate quantity of the contents comprising theinternal volume of the intake conduit, as described elsewhere herein. Ifthe volume of sample does not comprise air, it may be transferred to theprocess system by positioning the injector such that the common conduitis in fluid communication with the process system. If the volume ofsample comprises air, the sample may be rejected without transfer to theprocess system. In some embodiments, the sample is re-sampled to attemptto sample it without air. In other embodiments, intake of the sample isonly attempted once. In some embodiments, the aspiration tip is cleanedwith an outside wash collar to remove contaminants from the outer wallof the tip and/or blownback/injected with cleaning fluids internally toremove contaminants from the inner wall of the tip, as describedelsewhere herein. The embodiment shown in FIG. 17B is similar, but doesnot check whether the volume of sample comprises air.

FIG. 18—Systems for supplying sample fluids and cleaning fluids FIG. 18shows a system for supplying both cleaning fluids and sample fluids tobe aspirated. The system comprises a first set of fluid containers 1801with a first seal 1802 and a second set of fluid containers 1803 with asecond seal 1804. The first set of fluid containers comprise dispersedphase fluids for processing in the present system, and the second set offluid containers comprise cleaning fluids for processing in the presentsystem. In an embodiment shown in FIG. 18, the first set of fluidcontainers 1801 and the second set of fluid containers 1803 aremicrotiter plates. Microtiter plates can be any suitable size. Incertain embodiments, the plates are independently 24-well, 48-well,96-well, 384-well, or 1536-well microtiter plates.

FIG. 19—System for supplying cleaning fluids and FIG. 20—Self-fillingsystem for supplying cleaning fluids FIGS. 19 and 20 show a system forproviding cleaning fluids in the present system. The system comprises atleast one cleaning fluid reservoir 2001 that comprises a port 2002 andan internal volume 2003. An aspiration tip 2004 is positioned into thecleaning fluid reservoir 2001 through the port 2002, where it pushes ona surface 2005 and compresses a spring 2006 that opens a valve 2007,allowing cleaning fluid to pass through a cleaning fluid inlet 2008 intoa cleaning fluid conduit 2009. The cleaning fluid inlet 2008 isvertically positioned such that the volume of cleaning fluid allowed topass into the cleaning fluid reservoir 2001 is limited to that volume atwhich the vertical position of the surface of the cleaning fluid in thecleaning fluid reservoir 2001 is equal to the vertical position of thecleaning fluid inlet 2008. In certain embodiments, a first volume ofcleaning fluid may be aspirated while the valve 2007 is open, allowingadditional cleaning fluid to flow through the cleaning fluid inlet 2008.In certain embodiments, the first volume is zero. The aspiration tip2004 is positioned vertically such that the valve closes, and a portionof the cleaning fluid is aspirated into the aspiration tip 2004. Incertain embodiments, all of the cleaning fluid in the cleaning fluidreservoir 2001 is aspirated into the aspiration tip 2004. In certainembodiments, the valve is actuated by direct pressure of the end of theaspiration tip 2004 on the surface 2005. In other embodiments, the valveis actuated by pressure of an attachment to the outer wall of theaspiration tip 2004 on the surface 2005. In certain embodiments, thesystem additionally comprises a cover 2010 that may cover the cleaningfluid reservoir 2001 when it is not occupied by the aspiration tip 2004,reducing the possibility for environmental contamination. In certainembodiments, the cover 2010 is automatically actuated. In certainembodiments, the system additionally comprises at least one reservoir ofcleaning fluids vertically positioned above and in fluid communicationwith the cleaning fluid inlet 2008.

The sampling system may be positioned by mounting the carriage on aposition actuator. See e.g. FIG. 89. In some instances, the carriage islinearly positioned over a single dispersed phase reservoir comprisingsample (e.g. a PCR tube) or a linear array of single dispersed phasereservoirs comprising sample (e.g. a PCR strip). The reservoirs ofadditional dispersed phases may be positioned in the same linear array.The carriage may be moved back and forth and positioned over thedispersed phase to be injected. The sampling system may be actuated suchthat the sampling tube contacts the bottom of the dispersed phasereservoir for injection. The linear motion may be achieved using a leadscrew/lead nut combination. In some instances, the sampling systemcomprises physical stops, stop sensors, limit switches, or otherstopping mechanisms. In some instances, the sampling system runs in anopen loop. In some instances, a belt and pulley system or a linearactuator is used.

In some instances, the carriage comprises two degrees of positioningfreedom. The reservoirs of dispersed phase may comprise sample arearranged in a two-dimensional array (e.g. a microwell plate), and thereservoirs of additional dispersed phases are positioned so that thesampling system samples the additional dispersed phases in betweensampling the dispersed phase comprising sample. The positioning may beachieved by a pair of lead screws/lead nuts, a belt and pulley system,or a linear actuator. Exemplary belt and pulley systems include, but arenot limited to, H-bot pulley, direct belt drive, or Core XY pulley. Insome instances, the sampling device comprises a filter.

FIG. 21—Fluid aspirator FIG. 21 shows a system for aspirating a fluid.The system comprises an aspiration conduit 2010 with an aspiration tip2100, a seal piercer 2102, a first compliance spring 2103, a secondcompliance spring 2014, and an outer housing 2105. The seal piercer 2102has a conical end that concentrically surrounds the aspiration conduit2101 in a region near the aspiration tip 2100 and comprises a mechanicalstop surface 2106. To aspirate a fluid, the entire assembly isvertically actuated toward the top surface of a fluid container untilthe mechanical stop surface 2106 contacts an upper surface of the fluidcontainer. If the fluid container comprised a seal, the seal piercer2102 breaks the seal. At the point that the mechanical stop surface 2106contacts the upper surface of the fluid container, vertical motion ofthe seal piercer 2102 is arrested, but the first compliance spring 2103provides compliance to allow the aspiration conduit 2101 to continuevertical motion. In certain embodiments, vertical motion is stopped whenthe aspiration conduit 2101 is submerged in the fluid contents of thefluid container. In certain embodiments, vertical motion is not stoppeduntil the aspiration conduit 2101 contacts the bottom surface of thefluid container. Vertical motion of the outer housing may continue 2105without motion of the aspiration conduit 2101 due to the compliance ofthe second compliance spring 2104. Such embodiments remove therequirement that the vertical position of the bottom of the fluidcontainer be known to allow for maximal aspiration of fluid, avoidanceof mechanical damage to the aspirator tip, or both.

FIG. 22—System for aspirating fluid FIG. 22 shows a system foraspirating fluids. The system comprises at least one fluid container2201, a fluid container holder 2202, a first axis carriage 2203, a firstaxis drive 2204, a second axis carriage 2205, a second axis drive 2206,a vertical actuator 2207, and an aspiration conduit 2208. The fluidcontainer 2201 contains fluids to be aspirated. In some embodiments, thefluid contents of the fluid container 2201 comprise dispersed phase,e.g., samples as described further herein. The first axis drive 2204creates motion of the first axis carriage 2203 in a first axis and thesecond axis drive 2206 creates motion of the second axis carriage 2205.The second axis carriage holds the aspiration conduit 2208, and combinedmotion of the first axis drive 2204 and the second axis drive 2206 allowfor arbitrary positioning of the aspiration conduit 2208 on the planecreated by the first and second axes. In certain embodiments, the firstand second axis drives are a screw/nut combination turned by a motor. Incertain embodiments, the first and second axis drives are directlypositioned by belts. In a certain embodiments, movement of a first beltpositions the first axis carriage 2203 and movement of the second beltpositions the second axis carriage 2205. In certain embodiments, twobelts are arranged in an H-bot arrangement such that the combinedmotions of the two belts create movements of the first axis carriage2203 and the second axis carriage 2205 together. Motors may be anysuitable motors; in certain embodiments, the motors are DC brushedmotors, DC brushless motors, or stepper motors. Motors may be operatedin open-loop or closed-loop control. In certain embodiments, the systemadditionally comprises mechanical or electro-mechanical stops to preventpositioning the system beyond an allowable set of bounds. In certainembodiments, the system additionally comprises limit switches toindicate when the first or second axis positions of the carriages are atallowable limits. The limit switches may be any suitable switches; incertain embodiments, the limit switches are mechanical switches, opticalinterrupters, or proximity sensors. The vertical actuator 2207 moves theaspiration conduit 2208 in the vertical position, allowing it to beinserted into fluid containers for the purpose of aspirating fluids. Incertain embodiments, the vertical actuator comprises a linear actuatorand rotational motion-translation system. The vertical actuator may beany suitable actuator; in certain embodiments, the vertical actuatorcomprises a scotch yoke, rack-and-pinion, linear-actuator, orlinear-belt drive.

In certain embodiments, the system additionally comprises an air intake2209, an air outlet 2210, and an air fan 2211 for generating air flowbetween the air intake and air outlet. In certain embodiments, the airfan 2211 is closer to the air outlet 2210 than the air inlet 2209. Inother embodiments, the air fan 2211 is closer to the air inlet 2209 thanthe air outlet 2210. The airflow may be used to remove thermal energyfrom the underside of the fluid container holder 2202. In certainembodiments, the system further comprises a thermoelectric cooler suchthat the temperature of the fluid container 2201 may be held below amaximum temperature and heat from the thermoelectric cooler may beremoved by the airflow. In some embodiments, the maximum temperature isless than 27 C, less than 25 C, less than 21 C, less than 17 C, lessthan 15 C, less than 10 C, or less than 5 C. Maintaining a reducedtemperature in the fluid container 2201 may slow or prevent unwantedchemical or physical reactions in the fluid contents of the fluidcontainer 2201 prior to fluid aspiration.

FIG. 23—Fluid aspirator FIG. 23 shows a system for aspirating a fluidthat eliminates the need to precisely position an aspiration tip,comprising an aspiration conduit 2301, an aspiration tip 2302, amechanical counter-force surface 2303, a compliance spring 2304, and anouter housing 2305. The fluid aspirator is actuated into a fluidcontainer until the aspiration tip 2302 contacts the bottom surface ofthe fluid container. At this point, compliance provided by compressionof the compliance spring 2304 between the mechanical counter-forcesurface 2303 and the outer housing 2305 allows for continued travel ofthe outer housing 2305 without applying significantly increased forcesto the aspiration tip 2302. In certain embodiments, positioning of theaspiration tip 2302 at the bottom of a fluid container is important toaspirate as much of the fluid contents of the fluid container aspossible. This system reduces the need to know the vertical position ofthe aspiration tip 2302 accurately, as it may be guaranteed to be incontact with the bottom surface of the fluid container when thecompliance spring is compressed. In certain embodiments, the systemadditionally comprises a force sensor such that a signal from the forcesensor indicates when compression of the spring increases, allowing fordetection of when the aspiration tip 2302 is in contact with the bottomof the fluid container. In certain embodiments, the aspiration tip 2302is subsequently vertically positioned upward of the bottom of the fluidcontainer once the bottom surface has been located prior to aspiration.

FIG. 24—System for aspirating a fluid FIG. 24 shows a system foraspirating fluid comprising at least one fluid container 2401, a fluidcontainer holder 2401, a first axis carriage 2403, a first axis drive2404, a second axis carriage 2405, a second axis drive 2406, a firstvertical actuator 2407, and an aspiration conduit 2408. The fluidcontainer 2401 contains fluids to be aspirated. In some embodiments, thefluid contents of the fluid container 2401 comprise dispersed phase,such as samples, e.g., as described herein. The first axis drive 2404creates motion of the first axis carriage 2403 in a first axis and thesecond axis drive 2406 creates motion of the second axis carriage 2405.The second axis carriage holds the fluid container holder 2403, andcombined motion of the first axis drive 2404 and the second axis drive2406 allow for arbitrary positioning of the fluid container holder 2402on the plane created by the first and second axes. In certainembodiments, the fluid container holder may be positioned so that a usermay place a fluid container 2401 in the fluid container holder 2402. Incertain embodiments, the first and second axis drives are a screw/nutcombination turned by a motor. In certain embodiments, the first andsecond axis drives are directly positioned by belts. In certainembodiments, movement of a first belt positions the first axis carriage2403 and movement of the second belt positions the second axis carriage2405. In certain embodiments, two belts are arranged in an H-botarrangement such that the combined motions of the two belts createmovements of the first axis carriage 2403 and the second axis carriage2405 together. Motors may be any suitable motors; in certainembodiments, the motors are DC brushed motors, DC brushless motors, orstepper motors. Motors may be operated in open-loop or closed-loopcontrol. In certain embodiments, the system additionally comprisesmechanical or electro-mechanical stops to prevent positioning the systembeyond an allowable set of bounds. In certain embodiments, the systemadditionally comprises limit switches to indicate when the first orsecond axis positions of the carriages are at allowable limits. Anysuitable limit switches may be used; in certain embodiments, the limitswitches are mechanical switches, optical interrupters, or proximitysensors. The first vertical actuator 2407 moves the aspiration conduit2408 in the vertical position, allowing it to be inserted into fluidcontainers for the purpose of aspirating fluids. In certain embodiments,the vertical actuator comprises a linear actuator and rotationalmotion-translation system. Any suitable vertical actuator may be used;in certain embodiments, the vertical actuator comprises a scotch yoke,rack-and-pinon, linear-actuator, or linear-belt drive.

In certain embodiments, the system comprises a second vertical actuator2409 (FIG. 25). In certain embodiments, the system comprises a sealpunch 2510 positioned by the second vertical actuator 2509 such that theseal punch may pierce seals covering the at least one fluid container2501. In certain embodiments, the system comprises a second aspirationconduit 2511 positioned by the second vertical actuator 2509 such thatthe second aspiration conduit may aspirate fluid contents from ordispense fluid contents into the at least one fluid container 2501. Incertain embodiments, the system comprises both the seal punch 2510 andthe second aspiration conduit 2511.

In certain embodiments, the second aspiration conduit may aspirate atleast one cleaning fluid contents that have been dispensed into the atleast one fluid container 2501 by the first aspiration conduit 2508. Incertain embodiments, the second aspiration conduit aspirates at leastone cleaning fluid into a cleaning fluid waste reservoir.

In certain embodiments, the system additionally comprises an air intake2512, an air outlet 2513, and an air fan 2514 for generating air flowbetween the air intake and air outlet. In certain embodiments, the airfan 2514 is closer to the air outlet 2513 than the air inlet 2512. Incertain embodiments, the air fan 2514 is closer to the air inlet 2512than the air outlet 2513. The airflow may be used to remove thermalenergy from the underside of the fluid container holder 2502. In certainembodiments, the system further comprises a thermoelectric cooler suchthat the temperature of the fluid container 2501 may be held below amaximum temperature and heat from the thermoelectric cooler may beremoved by the airflow. In certain embodiments, the maximum temperatureis less than 27 C, less than 25 C, less than 21 C, less than 17 C, lessthan 15 C, less than 10 C, or less than 5 C. Maintaining a reducedtemperature in the fluid container 2501 may slow or prevent unwantedchemical or physical reactions in the fluid contents of the fluidcontainer 2501 prior to fluid aspiration.

FIG. 26—System for holding a fluid container FIG. 26 shows a system forholding a sample container comprising at least one fluid container 2601,a fluid container holder 2602, a thermoelectric cooler 2603, a heat sink2604, an airflow assembly 2605, and a fan 2606. The fluid container 2602holder has features such that the at least one fluid container 2601 mayonly be inserted into the fluid container holder 2602 in one way andthat thermal contact is maintained between the fluid container holder2602 and the at least one fluid container 2601. The fluid container canbe any suitable fluid container; in certain embodiments, the fluidcontainer is a microtiter plate, a tube strip, an individual test tube,or a cartridge. In certain embodiments, the fluid container is a 24-wellplate, a 48-well plate, a 96-well plate, a 384-well plate, or a1536-well plate. The thermoelectric cooler 2603 additionally comprises atemperature sensor and a temperature controller such that thetemperature of the fluid container 2601 may be maintained in a constanttemperature range. Any suitable method of control may be used; incertain embodiments, the temperature controller uses aproportional-integral method, a proportional-integral-differentialmethod, or an on-off method of control. Maintaining the temperature ofthe fluid container 2601 in a constant temperature range may bebeneficial to prevent biochemical or chemical reactions or physicalchanges to the fluid contents of the at least one fluid container 2601.In certain embodiments, the size of the temperature range is less than 5C, less than 3 C, less than 1 C, or less than 0.1 C and the constanttemperature is within the temperature range of 25 C, 20 C, 15 C, 10 C,or 5 C. The heat sink 2604 aids in heat transfer away from thethermoelectric cooler 2603 and the fan 2604 drives air through theairflow assembly to remove heat from the heat sink 2604.

In certain embodiments, the thermoelectric cooler 2603 may be run inreverse to add heat to the fluid contents of the at least one fluidcontainer 2601 to increase the temperature of the fluid contents to anelevated temperature in order to create a biochemical, chemical, orphysical change in the fluid contents of the at least one fluidcontainer 2601. In certain embodiments, the reaction is a cellularlysis, reverse transcription, nucleic acid polymerization, proteindenaturing, or any other suitable reaction. In certain embodiments, theelevated temperature is less than 96 C, less than 90 C, less than 80 C,less than 70 C. less than 60 C, or less than 50 C. In certainembodiments, multiple elevated temperatures are achieved.

FIGS. 27A, 27B, 27C, and 27D—System for cleaning a fluid aspirator FIG.27 shows a system for cleaning a fluid aspirator comprising a body 2701,an internal volume 2702, and a port 2703. A first fluid aspirator isinserted into the port 2703 and exposed to at least one cleaning fluid.In certain embodiments, the first fluid aspirator dispenses the at leastone cleaning fluid into the internal volume 2702. In certainembodiments, a second fluid aspirator dispenses the at least onecleaning fluid into the internal volume prior 2702 prior to insertion ofthe first fluid aspirator, and the external or internal surfaces of thefirst fluid aspirator may be cleaned by contact with or aspiration ofthe at least one cleaning fluid by the first fluid aspirator. In certainembodiments, a third fluid aspirator evacuates the at least one cleaningfluid from the internal volume 2702 after removal of the first fluidaspirator. In certain embodiments, the second and third fluid aspiratorare the same aspirator. In certain embodiments, the system additionallycomprises a drain 2703 for removal of the at least one cleaning fluid.In certain embodiments (FIGS. 27A and 27C) the system additionallycomprises a first cleaning fluid channel 2704 with a first cleaningfluid inlet 2705 that dispenses at least one cleaning fluid into theinternal volume 2702. In certain embodiments (FIGS. 27B and 27D), thesystem additionally comprises a second cleaning fluid channel 2706 witha second cleaning fluid inlet 2707. When the second cleaning fluid flowsfrom the second cleaning fluid inlet 2707 through the second cleaningfluid channel 2706 into the internal volume 2702, it impinges on the tipof the first fluid aspirator so as to improve removal of at least onefluid from the outer surface of the first fluid aspirator.

FIG. 28—System for providing sample and cleaning fluids FIG. 28 shows asystem for providing sample and cleaning fluids comprising at least onesample container 2801 and at least one cleaning fluid container 2802. Incertain embodiments, the at least one sample container is a microtiterplate and the at least one cleaning fluid container 2802 is an opentray. In certain embodiments, the microtiter plate is a 96-well plateand the system comprises two cleaning fluid containers 2802

FIG. 1 shows an intake system with a waste FIG. 1 shows an embodiment ofa system for conducting a process on one or more aliquots of samplefluid without cross-contamination of the sample fluids. The systemcomprises an intake system inlet 101, an autosampler assembly 102, aninjector 103, at least one waste 104, a process continuous fluid source105, and a process system inlet 106. The autosampler assembly 102 pullssample fluids into the injector through the intake system inlet 101 andpositions the fluids so that the process continuous fluid source 105 maydisplace the sample fluids into the process system inlet 106, asdescribed elsewhere herein. Once re-positioned to be in fluidcommunication with the intake system, cleaning fluids may be pulled intothe intake system inlet 101, displacing residual sample fluid into thewaste 104, with the composition, order and amount of cleaning fluids asdescribed elsewhere herein. The system may additionally comprise apartitioner 107 to sub-divide the aliquots of sample fluid, a reactor108 to mediate at least one reaction on the aliquots or partitions ofsample fluid, or a detector 109 to measure at least one property of thealiquots or partitions of sample fluid, or any combination thereof, eachas described herein.

FIG. 2 shows an intake system with blowback FIG. 2 shows anotherembodiment of the system in FIG. 1 comprising a sample inlet 201, anautosampler assembly 202, an injector 203, and where the waste 104 isreplaced by a source of purge or cleaning fluids 210. The system mayadditionally comprise a process continuous fluid source 205 and maycomprise a partitioner 207, a reactor 208, a detector 209, and/or aprocess waste 204, as described in FIG. 1. During the cleaning steps, asdescribed elsewhere herein, the purge or cleaning fluids are backflushedthrough the injector 203 and out the intake system inlet 201, and into awaste container, as described elsewhere herein.

FIG. 3 shows an intake system with spacer fluid partition FIG. 3 showsanother embodiment of the system where a source of spacer fluid 311 isadded on the process side of the system. The system comprises a sampleinlet 301, an autosampler assembly 302, an injector 303, and a processcontinuous fluid source 305. The system may additionally comprise apartitioner 307, a reactor 308, a detector 309, and/or a process waste304, as described in FIG. 1. In some embodiments, the source of spacerfluid is added after a partitioner 307, as shown. In other embodiments,the source of spacer fluid is added before or after the injector 303 butbefore the partitioner 307. In some embodiments, the source of spacerfluid may comprise a pump to move the spacer fluid into the processsystem. In some embodiments, the pump is actuated after a sample volumehas been transferred through the process system elements upstream of thespacer fluid source so as to add a spacer fluid volume after the samplevolume. In some embodiments, the pump is a metering pump, a positivedisplacement pump, a piston pump, a syringe pump, a diaphragm pump, or aperistaltic pump.

FIG. 29—System for providing fluid reagents FIG. 29 shows a system forproviding fluid reagents comprising a rigid cartridge 2901, acollapsible bag 2902, a fluid volume 2903, a fluid delivery conduit2904, a conduit coupling 2905 for connecting the fluid delivery conduit2904 to the collapsible bag 2902, and a cartridge interface 2906. Asfluid is delivered from the collapsible bag through the fluid deliveryconduit 2904, the collapsible bag collapses, eliminating the need tovent the fluid delivery conduit 2904 to account for the reduced fluidvolume. The rigid cartridge 2901 encloses the collapsible bag 2902,making it easier for users to handle. When the rigid cartridge 2901 iscoupled to the cartridge interface 2906, the conduit coupling 2905 issimultaneously coupled, and when the rigid cartridge 2901 is decoupledfrom the cartridge interface 2906, the conduit coupling 2905 issimultaneously decoupled, eliminating the need for the user to directlycouple and decouple the conduit coupling 2905, which may be difficult todo without introducing significant quantities of air into the system.

The conduit coupling can be any suitable coupling; in certainembodiments, the conduit coupling 2905 is a screw connector, a Luerconnector, a press quick-disconnect connector, or a screwquick-disconnect connector. In certain embodiments, the conduit coupling2905 has zero or very low dead volume. The collapsible bag may compriseany suitable material. In certain embodiments, the fluid deliveryconduit 2904 may be a tube. The tube may comprise any suitable material.In certain embodiments, the fluid delivery conduit 2904 may have aninternal volume capable of holding a minimum volume of fluid reagents.If each processing step requires an integral volume of fluid, the usermay not be able to use all of the fluid in the collapsible bag 2902because, at a point in time, an insufficient volume of fluid remains inthe collapsible bag 2902 to complete all of the processing steps. Bysizing the minimum volume such that the fluid delivery conduit 2904 mayhold enough fluid for a minimum number of processing steps, the user canmaximize usage of the fluid in the collapsible bag. In some embodiments,the minimum volume is greater than 1 mL, greater than 5 mL, greater than10 mL, or greater than 20 mL.

FIG. 30. FIG. 30 shows a further embodiment where the rigid cartridgeencloses a plurality of collapsible bags, each connected to a fluidconduit. This embodiment allows for the user to install multiple reagentreservoirs with a single replaceable cartridge. FIG. 30 showsembodiments of a multi-container cartridge for supplying reagents orcollecting waste. The system comprises a cartridge 3001 to holds atleast one fluid container 3002 with a fluid container line 3003 toconnect to a primary fluid container connector 3004. In someembodiments, the fluid container 3002 comprises materials that areflexible, such that the volume of the fluid container 3002 may decreaseas fluid is dispensed or increase as fluid is collected. The fluidcontainer 3002 may be comprised of any suitable materials. In someembodiments, the fluid container comprises a polymer or a metal. Thefluid container line 3003 may be comprised of any suitable material. Insome embodiments, the fluid container line 3003 comprises a polymer or ametal. The cartridge 3001 may comprise a vent hole to allow air to enteror leave the cartridge as the volume of the at least one primary fluidcontainer 3002 increases or decreases. The primary fluid containerconnector 3004 interfaces to a secondary fluid container connector 3005that interfaces with one or more conduits on the intake system and/orprocess system. In some embodiments, the primary fluid containerconnector 3004 and secondary fluid container connector 3005 are quickdisconnect connectors. In some embodiments, the primary fluid containerconnector 3004 and secondary fluid container connector 3005 aredry-break connectors so as to minimize fluid leak or loss whenconnecting or disconnecting the cartridge 3001. In some embodiments, thefluid container 3002 additionally comprises one or more additionaloutlets or vents to allow for draining or addition of fluid to thecontainer. In some embodiments, the cartridge 3001 holds two or morefluid containers, each with a fluid container line, a primary fluidconnector, and a secondary fluid connector, such that a user may connectall of the fluid connectors simultaneously when connecting any one fluidconnector. In some embodiments, the fluid containers comprise reagentspre-loaded for use in the intake or process systems. In someembodiments, the one or more fluid containers are for collecting waste.In some further embodiments, the one or more fluid containers comprise adenaturing fluid for denaturing at least one detectable or potentiallydetectable component that may be added to the fluid containers 3002 inthe operation of the intake and/or process system.

Thus, an external reservoir system may be used for systems and methodsas described herein. The external reservoir may comprise a polymer bag,a tubular channel, or a luer-lock fitting, or other materials orcomponents as described herein. In some instances, the polymer bag is acollapsible polymer bag. In some instances, the collapsible polymer bagallows fluid to flow by gravity into the reservoir as needed without airexchange back into the bag. In some instances, the polymer bag is placedinside a hard-sided bottle. The hard-sided bottle may have a threadedinterface at the bottom that holds the luer-fitting for the polymer bagin a fixed position. In some instances, a mating threaded interfaceabove the reservoir holds a mating luer-fitting in a fixed position.

FIG. 31 (syringe pump) FIG. 31 shows an embodiment of a system forpumping fluid phases through conduits within the system. The systemcomprises a motor 3101 that is coupled to a lead screw 3102, such thatrotational motion from the motor is transferred to motion of the leadscrew. A lead nut 3103 is threaded over the lead screw and is coupled toa piston 3104, that is mounted inside of a bore or cavity. As the leadscrew rotates the piston travels along the lead screw, either towardsthe motor as the motor spins in one direction and away from the motor asit spins in the opposite direction. As the piston moves through the boreit either pulls fluid into the bore or pushes fluid out of the bore. Thebore has inlet 3106, and outlet 3107 ports that can couple to tubing orother system components such as fluid reservoirs. The substrate of thebore may be comprised of any suitable material that is chemicallyresistant to the system fluids contained within the syringe pump.

In certain embodiments the system contains an inlet check valve at theinlet port and an outlet check valve at the outlet port. These checkvalves are oriented such that the inlet check valve will be opened, andthe outlet check valve closed when the piston is being driven towardsthe motor such that fluids will flow into the bore from through inletand will not exit the outlet. While the piston is being driven away fromthe motor the inlet check valve will seat closed and the outlet checkvalve will crack open allowing fluid to be pushed. In certainembodiments, the inlet to the syringe pump is connected to a reagentsupply reservoir. In certain embodiments, the inlet port of the syringepump is connected to a switching valve that is also connected tomultiple reagent reservoirs such that the switching valve can positionitself to allow one of multiple reagent reservoirs to feed into thesyringe pump.

The motor may be any suitable motor; in certain embodiments the motor isa stepper motor, such a motor might be controlled such that it moves inmicro stepping mode. In certain embodiments each micro step translatesto a fluid displacement that is, e.g. 1%, 2%, 5%, 10%, 25%, 50%, 100%,200% the size of the dispersed phase partitions that are created by thesystem partitioner. In certain embodiments a gear box is coupled betweenthe motor shaft and the lead screw. In certain embodiments the motor isa brushed DC motor, or a brushless DC motor, or a servo motor.

In some embodiments an encoder is included as part of the assembly suchthat it can provide feedback to the rotation of the motor shaft. Theencoder output can be used to help control the speed of the motorrotation, providing control over the rate of fluid flow into and out ofthe syringe pump. In certain embodiments, the fluids are pumped throughthe system not with a positive displacement pump, but with a pneumaticsystem where a pressure head is created to drive fluids to desiredlocations.

Further components can include one or more of a limit switch to home orfind absolute position, an encoder to store memory of absolute position,an anti-backlash nut, various connector styles, orientation to avoid airbeing pulled into the syringe pumps, o-rings, a flag to preventrotation, materials, multiway valves such as 3 way valves. In certainembodiments pump is configured to provide nonpulsatile flow, such thatthe flow is consistent enough to drive droplet formation where thecoefficient of variation of droplet size is less than 5%, 4%, 3%, 2%,1%.

In certain embodiments, 2 or more syringe pumps are contained within thesystem to drive the same system fluid such that if either pump fails thesystem can still dispense said system fluid. In certain embodimentspumps can be controlled such that one pump will stop dispensing while asecond pump starts dispensing the same fluid into towards the samechannel such that the fluid flow is not disrupted for more than 10 ms,100 ms, 1 s, 5 s, 10 s or 20 seconds.

FIG. 32 (bank of syringe pumps) In some embodiments at least 2, 3, 4, 5,6, 7, 8, 9, or 10 syringe pumps are included in the system, e.g., todrive different system fluids and deliver them to components of thesystem. The design of the syringe pump allows large numbers ofindividual pumps to be arranged without taking up significant amount ofspace that would drive an increase in the overall system footprint

Thus, described herein are systems and methods for serial flow emulsionreactions comprise use of a sampling device, wherein the sampling devicecomprises a pump. The pump may be used to provide a driving force tomove fluid from the fluid injector, the decontamination fluid reservoir,the purge fluid reservoir, or combinations thereof into the injectiondevice. See e.g. FIGS. 87-88. The pump may be located upstream ordownstream of the injector. In some instances, the pump is a peristalticpump. In some instances, the pump is reversible In some instances, thepump comprises a fluid channel that provides a physical barrier betweenmechanical elements of the pump and the working fluids of the system. Insome instances, a pump motor is actuated by a controller to move a setvolume of fluid from the fluid injector, fluid reservoir, purge fluidreservoir, or combinations thereof into the injection device. In someinstances, the pump motor is a stepper motor. In some instances, thepump motor is a servo motor with a rotational encoder. In someinstances, the controller operates in an open-loop mode. In someinstances, the controller operates in closed-loop mode.

Thus, systems and methods as described herein may comprise a pump. Thepump may be downstream of the fluid injector, decontamination fluidreservoir, and purge reservoir but upstream of the injection device. Thepump may create suction in the fluid injector, decontamination fluidreservoir, or purge reservoir, drawing fluid from these elements andcreating a positive pressure downstream that drives the fluid into theinjection device. In some instances, the pump is downstream of the fluidinjector, decontamination fluid reservoir, or purge reservoir, and theinjection device. The pump may create suction throughout both thesampling device and injection device to draw fluid through both devices,which is deposited in a waste reservoir.

In some instances, the sampling device comprises a flush reservoir. Theflush reservoir may comprise a fluid immiscible with a dispersed phasethat forms a dispersed phase in the microfluidic channel. In someinstances, the fluid contained in the flush reservoir comprisesmaterials at least partially miscible with a dispersed phase comprisingsample. In some instances, the fluid contained in the flush compriseswater and PCR primers and fluorescent labels for a biological assaybeing performed on nucleic acids in the dispersed phase comprisingsample. In some instances, the dispersed phase does not comprise nucleicacids and is used as a “no template control.” In some instances, theflush comprises fluorescent markers to indicate a boundary betweensamples.

III. Injector

Systems and methods provided herein can include an injector, alsoreferred to herein as an injection system. In certain embodiments, aninjector serves as an interface between the intake system, also referredto herein as a sampler, autosampler, or similar wording, and the processsystem, where the injector can cycle between a configuration that is influid communication with the intake system and a configuration that isfluid communication with the process system. The injector can beconfigured to have additional configurations, e.g., configurations thatallow cleaning of one or more parts of the intake system and injector.In certain embodiments, the injector comprises common conduit, alsoreferred to herein as an injection chamber, or injection loop, where thecommon conduit can be in fluid communication with the intake system orin fluid communication with the process system, but cannotsimultaneously be in fluid communication with both. Thus, the intakesystem and/or the injector can be treated between injections ofdispersed phase, e.g., between samples, in order to reduce or eliminatetraces of dispersed phase, e.g., sample, between rounds of intake ofdispersed phase, e.g., sample. It will be appreciated that “intakesystem” can include the injector when the system is configured to befluidly connected to the injector, as will be clear from context in thefollowing description.

In certain embodiments, systems and methods provided herein may comprisean intake system, such as any of the intake systems described above,connected to an injector in such a way that in a first configurationthere is fluid communication between the injector and the intake systemand in a second configuration there is not fluid communication betweenthe injector and the intake system. In further embodiments, in thesecond configuration the injector is in fluid communication with aprocess system. The process system can comprise a partitioner, such as apartitioner as described below. The process system can comprise areactor, such a reactor as described below. The process system cancomprise a detector, such as a detector as described below. The processsystem can comprise a disengager, such as a disengager as describedbelow. In certain embodiments, the process system comprises apartitioner and a reactor. In certain embodiments, the process systemcomprises a partitioner, a reactor, and a detector. In certainembodiments, the process system comprises a partitioner, a reactor, adetector, and a disengager. In certain embodiments, the process systemis a system for performing a digital process in partitions, such as adigital PCR system. In certain embodiments, surfaces of the injectorthat come in contact with a dispersed phase, such as a sample, havehigher affinity for at least a first continuous phase than for thedispersed phase; in certain embodiments, surfaces of the injector thatcome in contact with dispersed phase, e.g., sample, comprisefluoropolymer and the at least first continuous phase comprises afluorinated oil.

FIG. 33—Basic injector concept FIG. 33 shows an embodiment of aninjector. The injector comprises a common conduit 3301, a first intakesystem conduit 3302, a second intake system conduit 3303, a firstprocess system conduit 3304, and a second process system conduit 3305.The injector may be positioned in at least two states. In the firststate, the common conduit 3301 is in fluid communication with both thefirst intake system conduit 3302 and the second intake system conduit3303. In the second state, the common conduit 3301 is in fluidcommunication with both the first process system conduit 3304 and thesecond process system conduit 3305. The injector may exist in at mostone of these states at any given time. The injector may exist in neitherof these states, for example while the injector is changing between thefirst state and the second state or vice versa. As such, the injectorcan never be positioned such that the first intake system conduit 3302or the second intake system conduit 3303 are in fluid communication witheither the first process system conduit 3304 or the second processsystem conduit 3305. In general, the common conduit 3301, first intakesystem conduit 3302, second intake system conduit 3303, first processsystem conduit 3304, and second process system conduit 3305 may beconstructed of any suitable material. In certain embodiments, theconduits have surfaces that comprise a material that has a higheraffinity for at least one continuous phase in the system than for anydispersed phase in the system, such that when the at least onecontinuous phase flows through the system it preferentially displacesvolumes of dispersed phase from the surface of the conduit. In certainembodiments, the surface of the conduits in the system comprise ahydrophobic material, at least one continuous phase comprises ahydrophobic component, and at least one dispersed phase comprises anaqueous phase. In certain embodiments, the surface of the conduits inthe system comprises a fluoropolymer, at least one continuous phasecomprises a fluorinated oil, and the dispersed phase has a loweraffinity for a fluoropolymer surface than the fluorinated oil. Here andelsewhere herein, a fluoropolymer may be any suitable fluoropolymer,such as polytetrafluoromethylene (PTFE), chlorotrifluoroethylene (CTFE),polyvinylidene difluoride (PVDF), perfluoroalkoxy polymer (PFA),fluorinated ethylene-propylene (FEP), or a combination thereof. In someembodiments, the surface of the conduits comprises a hydrophilicmaterial, at least one continuous phase is hydrophilic, and at thedispersed phase is hydrophobic. In a further embodiment, the dispersedphase is an oil.

The common conduit 3301 may be constructed in any suitable manner. Incertain embodiments, the common conduit 3301 comprises a tube. Incertain embodiments, the common conduit 3301 comprises a fluoropolymertube. In certain embodiments, the common conduit 3301 comprises achannel in a solid substrate. In certain embodiments, the channel is amicrofluidic channel. In some further embodiments, the substratecomprises a fluoropolymer material. In some embodiments, the commonconduit 3301 comprises both a channel in a solid substrate and a tube.In some embodiments, the volume of the common conduit 3301 is selectedso as to deliver a specific aliquot of volume from the intake system tothe process system. This volume may be any suitable volume, such as avolume between 0.1 uL and 500 uL, or between 0.1 and 200 uL, or between0.5 and 200 uL, or between 1 and 200 uL, or between 2 and 200 uL, orbetween 2 and 100 uL, or between 2 and 50 uL, or between 2 and 30 uL, orbetween 5 and 200 uL, or between 5 and 100 uL, or between 5 and 50 uL,or between 5 and 40 uL, or between 5 and 30 uL, or between 10 and 200uL, or between 10 and 100 uL, or between 10 and 50 uL, or between 10 and30 uL.

Use of the injector system may allow for the movement of a specificvolume of a dispersed phase from the intake system to the processsystem. In certain embodiments, a method for moving a specific volume ofa dispersed phase from the intake system to the process system comprisespositioning the injector such that the common conduit 3301 is in fluidcommunication with the first intake conduit 3302 and the second intakeconduit 3303, flowing a dispersed phase through the first intake conduit3302, into the common conduit 3301, and at least partially into thesecond intake conduit 3303. The injector is then positioned such thatthe common conduit is in fluid communication with the first processconduit 3304 and the second process conduit 3305. At least onecontinuous phase is flowed into the first process conduit 3304,displacing the dispersed phase from the common conduit 3301 and into thesecond process conduit 3305. A volume of dispersed phase substantiallyequal to the volume of the common conduit 3301 less at most the volumeof a thin film of continuous phase near the surface of the commonconduit 3301 is then transferred from the intake side to the processside. In some embodiments, this process is repeated to transfer multiplevolumes of dispersed phase from the intake side of the system to theprocess side of the system, such as a series of at least 2, 5, 10, 50,100, 200, 500, or 1000 separate volumes of dispersed phase.

In certain embodiments, the common conduit 3301 has a higher affinityfor the at least one continuous phase than for the dispersed phase. Forexample, the common conduit can have a surface that is a fluoropolymerand the continuous phase can comprise a fluorinated oil. Thus, when atleast one continuous phase is flowed through the first process conduit3304 into the common conduit 3301, it preferentially displaces dispersedphase from the common conduit 3301 into the second process conduit 3305.

In embodiments where the common conduit 3301, first intake conduit 3302,first process conduit 3304, and the second intake conduit 3305 have ahigher affinity for at least one continuous phase than for the dispersedphase to be transferred from the intake system to the process system,the injector may also be used to transfer a plurality of volumes of atleast one dispersed phase from the intake system to the process system,such as a series of at least 2, 5, 10, 50, 100, 200, 500, or 1000separate volumes of dispersed phase, without cross-contamination betweenvolumes of the dispersed phase or phases, or with substantially nocross-contamination. Certain embodiments of the method comprisepositioning the common conduit 3301 such that it is in fluidcommunication with the first intake conduit 3302 and the second intakeconduit 3303, flowing a first volume of a dispersed phase through thefirst intake conduit 3303 such that flows into the common conduit 3301,positioning the common conduit 3301 such that it is in fluidcommunication with the first process conduit 3304 and the second processconduit 3305, flowing at least one continuous phase through the firstprocess conduit 3304 into the common conduit 3301 such that the at leastone continuous phase displaces or substantially displaces the firstvolume of dispersed phase from the common conduit 3301 into the secondprocess conduit 3304, positioning the common conduit 3301 such that itis in fluid communication with the first intake conduit 3302 and secondintake conduit 3303, flowing at least one continuous phase into thefirst intake conduit 3302 and through the common conduit 3301 such thatit displaces or substantially displaces the residual volume of the firstvolume of dispersed phase into the second intake conduit 3303, flowing asecond volume of dispersed phase through the first intake conduit 3302into the common conduit 3301, positioning the common conduit 3301 suchthat it is in fluid communication with the first process conduit 3304and the second process conduit 3305, and flowing at least one continuousphase through the first process conduit 3304 such that it displaces orsubstantially displaces the second volume of dispersed phase into thesecond process conduit 3305. In certain embodiments, the fraction of thefirst volume of dispersed phase or the second volume of dispersed phasedisplaced into the second process conduit 3305 is at least 80%, at least90%, at least 95%, at least 98%, at least 99%, at least 99.9%, at least99.99%, or at least 99.999%. In certain embodiments, the fraction of theresidual volume of the first volume of dispersed phase or second volumeof dispersed phase displaced into the second intake conduit 3303 is atleast 80%, at least 90%, at least 95%, at least 98%, at least 99%, atleast 99.9%, at least 99.99%, or at least 99.999%. As such, the commonconduit 3301 and portions of the intake system that were exposed to thefirst volume of dispersed phase are cleaned or substantially cleaned ofthe first volume of dispersed phase so that the second volume ofdispersed phase will not be contaminated with the first volume ofdispersed phase in the intake system, common conduit 3301, or processsystem. In certain embodiments, the first intake conduit 3302 comprisesa sample source and at least one continuous phase source and the secondintake conduit 3303 comprises a waste. In certain embodiments, the firstintake conduit 3302 may be positioned to be in fluid communication witha sample container and the second intake conduit 3303 comprises a motiveforce source. In certain embodiments, instead of flowing at least onecontinuous phase into the first intake conduit 3302 to displace residualvolume of the first or second volume of dispersed phase into the secondintake conduit 3303, at least one continuous phase may be flowed intothe second intake conduit 3303 through the common conduit 3301 todisplace residual dispersed phase into the first intake conduit 3302. Insuch embodiments, at least part of the second intake conduit 3303comprises a material that has a higher affinity for the at least onecontinuous phase than for the dispersed phases. In certain embodiments,the second intake conduit 3303 comprises a continuous phase source andthe first intake conduit comprises a sample source and a waste. Incertain embodiments, the first intake conduit 3302 comprises a samplesource and a waste.

In the above embodiments, a fixed volume of each sample may betransferred from the intake system to the process system if, whenflowing each sample through the first intake conduit 3302 into thecommon conduit 3301, the sample substantially fills the common conduit3301 (less the volume of a thin layer of at least one continuous phaseat the surfaces of the common conduit 3301) and partially into thesecond intake conduit 3303 such that, when the common conduit 3301 ispositioned to be in fluid communication with the first process conduit3304 and the second process conduit 3305, the volume of the commonconduit 3301 (less the volume of a thin layer of at least one continuousphase at the surfaces of the common conduit 3301) is transferred betweenthe intake system and the process system. This may be useful whereprecise aliquots of samples are to be transferred between the intakesystem and the process system. Alternatively, in some embodiments thecommon conduit 3301 may be partially filled with dispersed phase, withthe balance of the volume of the common conduit 3301 filled with atleast one continuous phase, before positioning the common conduit 3301so that that common conduit 3301 is in fluid communication with thefirst process conduit 3304 and the second process conduit 3305. This maybe useful where it is important to transfer a high fraction of eachvolume of sample between the intake system and the process system.

The above methods may be repeated any number of times to transfermultiple aliquots of one or more dispersed phases from the intake systemto the process system. In the above embodiments, there may be moments(e.g. while the common conduit 301 is transitioning between states)where there is no flow. As such, the system may be thought of as usingdiscontinuous flow to transfer aliquots of dispersed phases between theintake system and the process system. In certain embodiments, there is atransition pause in flow as the common conduit transitions betweenstates of at least 0.01, 0.05, 0.1, 0.5, 1, 2, 5, or 10 ms, and/or notmore than 0.05, 0.1, 0.5, 1, 2, 5, 10 or 50 ms.

FIG. 34—Injector with a plurality of common conduits FIG. 34 shows anembodiment of the injector where there is a plurality of commonconduits. The system comprises a first common conduit 3401 and a secondcommon conduit 3402, a first intake system conduit 3403, a second intakesystem conduit 3404, a first process system conduit 3405, and a secondprocess system conduit 3406. Each common conduit has at least twostates. In a first state of the first common conduit 3401, the firstcommon conduit 3401 is configured such that it is in fluid communicationwith the first intake conduit 3403 and the second intake conduit 3404.In a second state of the first intake conduit 3401, the system isconfigured such that the first common conduit 3401 is in fluidcommunication with the first process conduit 3405 and the second processconduit 3406. The first common conduit 3401 may be in at most one of itsfirst or second states at any given time, and the first common conduit3401 may be in neither of the states (e.g. when it is changing betweenstates). Likewise, the second common conduit 3402 has a first statewhere the second common conduit 3402 is positioned such that the secondcommon conduit 3402 is in fluid communication with the first intakeconduit 3403 and the second intake conduit 3404, and the second commonconduit 3402 has a second state where the second common conduit 3402 isin fluid communication with the first process conduit 3405 and thesecond process conduit 3406. The second common conduit 3402 may be in atmost one of its first and second states at any time, and it may be inneither of the first or second states (e.g. when it is changing betweenstates). The first common conduit 3401 and the second common conduit3402 may not simultaneously be in their respective n^(th) state; forexample, the first common conduit 3401 may not be in its first statewhile the second common conduit 3402 is in its first state, and thefirst common conduit 3401 may not be in its second state when the secondcommon conduit 3402 is in its second state. However, other states arenot mutually exclusive. Specifically, the first common conduit 3401 maybe in its first state when the second common conduit 3402 is in itssecond state, and the first common conduit 3401 may be in its secondstate when the first common conduit 3402 is in its first state.

In further embodiments, the first common conduit 3401 is in its firststate whenever the second common conduit 3402 is in its second state andthe first common conduit 3401 is in its second state whenever the secondcommon conduit 3402 is in its first state. Methods for efficientlytransferring volumes of at least one dispersed phase from the intakesystem to the process system can comprise positioning the first commonconduit 3401 such that it is in its first state (i.e. it is in fluidcommunication with the first intake conduit 3403 and the second intakeconduit 3404); the second common conduit 3402 will then be in its secondstate. A first volume of dispersed phase is flowed through the firstintake conduit 3403 and into the first common conduit 3401, and thefirst common conduit is positioned such that it is in its second state(simultaneously positioning the second common conduit 3402 in its firststate). At least one continuous phase is flowed through the firstprocess conduit 3405 and into the first common conduit 3401 such that itdisplaces all or a substantial fraction of the first volume of dispersedphase from the first common conduit 3401 and into the second processconduit 3406. While the at least one continuous phase is flowing throughthe first process conduit 3405, at least one continuous phase is flowedeither through the first intake conduit 3403, into the second commonconduit 3402, and into the second intake conduit 3404, displacing orsubstantially displacing residual dispersed phase into the second intakeconduit 3404, or through the second intake conduit 3404, into the secondcommon conduit 3402 and into the first intake conduit 3403, displacingor substantially displacing residual dispersed phase into the firstintake conduit 3403. A second volume of dispersed phase is then flowedthrough either the first intake conduit 3403 or second intake conduit3404 into the second common conduit 3402. Once the substantial fractionof the first volume of dispersed phase is displaced from the firstcommon conduit 3401 and the second volume of dispersed phase is flowedinto the second common conduit 3402, the injector is positioned suchthat the first common conduit 3401 is in its first state (simultaneouslyputting the second common conduit 3402 in its second state). At leastone continuous phase is flowed through the first process intake 3405 andinto the second common conduit 3402, displacing the second volume ofdispersed phase or a second substantial fraction of the second volume ofdispersed phase into the second process intake 3406. While the secondvolume of dispersed phase is being displaced into the second processintake 3406, at least one continuous phase is flowed through the firstintake conduit 3403 and into the first common conduit 3401, displacingresidual first volume of dispersed phase into the second intake conduit3404, or at least one continuous phase is flowed through the secondintake conduit 3404 and into the first common conduit 3401, displacingresidual first volume of dispersed phase into the first intake conduit3404. The first common conduit 3401 is then ready for intake of asubsequent volume of dispersed phase without cross-contamination of thedispersed phase volumes. These embodiments allow for cleaning of onecommon conduit while transferring volume from the second common conduitto the process system, reducing the amount of time required for a singlevolume of dispersed phase from the intake system to the process systemwithout cross contamination.

FIG. 35—Multi-position injector FIG. 35 shows an injector system thathas more than two states for a common conduit. The system comprises acommon conduit 3501, a first intake system conduit 3502, a second intakesystem conduit 3503, a first process system conduit 3504, a secondprocess system conduit 3505, a first auxiliary system conduit 3506, anda second auxiliary system conduit 3507. The common conduit 3501 has atleast three states. In the first state, the common conduit 3501 is influid communication with the first intake system conduit 3502 and thesecond intake system conduit 3503. In the second state, the commonconduit 3501 is in fluid communication with the first process systemconduit 3504 and the second process system conduit 3505. In the thirdstate, the common conduit 3501 is in fluid communication with the firstauxiliary system conduit 3506 and the second auxiliary system conduit3507. The system may be in at most one state at any time. This conceptmay be extended to systems with two, three, four, or any number ofauxiliary systems.

FIGS. 36A and 36B—Rotary single face injector FIG. 36 shows anembodiment of an injector system that comprises a rotary surface seal.The system comprises a first common conduit 3601 that comprises a firsttube, a second common conduit 3602 that comprises a second tube, a firstintake conduit 3603, a second intake conduit 3604, a first processconduit 3605, and a second process conduit 3606. The system additionallycomprises a first substrate 3607 which comprises a first port 3608, asecond port 3609, third port 3610, a fourth port 3611, a fifth port3612, sixth port 3613, a seventh port 3614, and an eighth port 3615. Thesystem additionally comprises a second substrate 3616 which comprises afirst channel 3617, a second channel 3618, a third channel 3619, and afourth channel 3620. The second substrate 3616 has at least two states,and the second substrate 3616 is moved between states by changing therelative rotational orientation of the first substrate 3607 and thesecond substrate 3616. In the first state, the first channel (FIG. 36A)3617 is in fluid communication with the first port 3608 and the secondport 3609, the second channel 3618 is in fluid communication with thethird port 3610 and the fourth port 3611, the third channel 3619 is influid communication with the fifth port 3612 and the sixth port 3613,and the fourth channel 3620 is in fluid communication with the seventhport 3614 and the eight port 3615. In the second state (FIG. 36B), thefirst channel 3617 is in fluid communication with the first port 3608and the sixth port 3613, the second channel 3518 is in fluidcommunication with the fourth port 3511, the third channel 3619 is influid communication with the third port 3610 and the fifth port 3612,and the fourth channel 3620 is in fluid communication with the secondport 3609 and the eighth port 3615. The first intake conduit 3603 is influid communication with the first port 3608, the second intake conduit3604 is in fluid communication with the fourth port 3611, the firstprocess conduit 3605 is in fluid communication with the fifth port 3612,and the second process conduit 3606 is in fluid communication with theeight port 3615. The first common conduit 3601 additionally comprises afirst conduit portion in fluid communication with the second port 3609and the sixth port 3613, and the second common conduit 3602 additionallycomprises a second conduit portion in fluid communication with the thirdport 3610 and the seventh port 3614. Thus, when the second substrate3616 is in the first state, the first common conduit 3601 is in fluidcommunication with the first intake conduit 3603 and the second intakeconduit 3604, and the second common conduit 3602 is in fluidcommunication with the first process conduit 3605 and the second processconduit 3606. When the second substrate 3616 is in the second state, thefirst common conduit 3601 is in fluid communication with the firstprocess conduit 3605 and the second process conduit 3606 and the secondcommon conduit 3602 is in fluid communication with the first intakeconduit 3603 and the second intake conduit 3604. When the relativeposition of the first substrate 3607 and the second substrate 3616 issuch that the second substrate 3616 is not in either state, the firstcommon conduit 3601 and the second common conduit 3602 are not in fluidcommunication with either the intake system or the process system.

The first substrate 3607 and second substrate 3616 may be made of anysuitable material. In some embodiments, the surfaces of the firstsubstrate 3607 and/or second substrate 3616 have a higher affinity forat least one continuous phase than for at least one dispersed phase inthe present system. In some embodiments, the surfaces of the firstsubstrate 3607 and/or second substrate 3616 comprise fluorinatedpolymers, and at least one continuous phase comprises a fluorinated oil,as described herein. In some embodiments, the first substrate 3607 ismoved relative to the second substrate 3616. In some embodiments, thesecond substrate 3616 is moved relative to the first substrate 3607.Motion can be achieved by any suitable mechanism. In certainembodiments, motion is achieved by using a motor. Any suitable motor maybe used, such as a stepper motor, a brushed dc motor, or a brushless dcmotor. In certain embodiments, the motor is a stepper motor and angularposition is determined using open-loop control by counting the number ofsteps moved. In certain embodiments, the motor has an encoder fordetermining the angular position. In certain embodiments, the systemcomprises limit switches to detect an angular position.

FIG. 37 Rotary dual face injector FIG. 37 shows an injector system thatcomprises two faces. The system comprises a first substrate 3701comprising at least one common conduit 3702. The system additionallycomprises a second substrate 3703 comprising a first port 3704 and asecond port 3705; third substrate 3706 comprising a first port 3707 anda second port 3708; a first intake conduit 3709 and second intakeconduit 3710; and a first process conduit 3711 and second processconduit 3712. The first intake conduit 3709 is in fluid communicationwith the first port 3704 of the second substrate 3703, the second intakeconduit 3710 is in fluid communication with the first port 3707 of thethird substrate 3706, the first process conduit 3711 is in fluidcommunication with the second port 3705 of the second substrate 3704,and the second process conduit 3712 is in fluid communication with thesecond port 3708 of the third substrate 3706. The common conduit 3702may exist in at least two states. In the first state, the common conduit3702 is in fluid communication with the first port 3704 of the secondsubstrate 3703 and the first port 3707 of the third substrate 3706, andis thus in fluid communication with the intake system. In the secondstate, the common conduit 3702 is in fluid communication with the secondport 3705 of the second substrate 3703 and the second port 3708 of thethird substrate 3706, and is thus in fluid communication with theprocess system. The common conduit 3702 may exist in at most one ofthese states at any time, although it may not exist in either state,e.g. when changing between the states. In some embodiments, the commonconduit 3702 is a channel or bore through a solid substrate. In someembodiments, the common conduit 3702 and ports 3704, 3705, 3706, and3708 are arranged such that changes in relative rotational position ofthe first substrate 3702 and the second and third substrates (3703 and3706, respectively) may position the common conduit 3702 in either ofthe states. In some embodiments, the first substrate 3702 is rotated andthe second substrate 3703 and third substrate 3706 are held in a staticposition. As described for other embodiments of the injector, the systemmay have multiple common conduits with mutually exclusive states. Insome embodiments, multiple common conduits are formed from channels in afirst substrate 3702 whose centerlines lie on a common circular arc.Motion may be generated in substrate 3702 (or substrates 3703 or 3706)by the use of a motor. In some embodiments, the motor is a brushed dcmotor, a stepper motor, a brushless dc motor. Position may be indicatedby any appropriate methods. In some embodiments, step counts, encoders,limit switches, and/or optical interrupters are used to determinepositions. The substrates 3702, 3703, and 3706 may be made of anysuitable material. In some embodiments, as in other descriptions ofinjectors herein, the surfaces of the substrates in contact with fluidshave a higher affinity for at least one continuous phase than for thedispersed phases in the system so as to allow preferential displacementof dispersed phases. In some embodiments, the substrates 3702, 3703, or3706 comprise metals or polymers. In some preferred embodiments, thesubstrates 3702, 3703, or 3706 comprise fluoropolymers and at least onecontinuous phase comprises a fluorinated oil. In further embodiments,the substrates 3702, 3703, or 3706 comprise CTFE and/or PTFE.

Thus, the sampling device may introduce volumes of dispersed phase to aninjector that then flows to a reactor and a detector. Injectors for usein systems and methods as described herein may comprise zero or low-deadvolume valves. In some instances the zero or low-dead volume valvescomprise one or more rotors. In some instances, one or dispersed phasespass through the rotor and injected through the injector. In someinstances, the rotor comprises a calibrated volume. In some instances,the calibrated volume is at least or about 0.001 nanoliter (nL), 0.002nL, 0.003 nL, 0.004 nL, 0.005 nL, 0.006 nL, 0.007 nL, 0.008 nL, 0.009nL, 0.01 nL, 0.02 nL, 0.03 nL, 0.04 nL, 0.05 nL, 0.06 nL, 0.07 nL, 0.08nL, 0.09 nL, 0.10 nL, 0.20 nL, 0.30 nL, 0.40 nL, 0.50 nL, 0.60 nL, 0.70nL, 0.80 nL, 0.90 nL, 1.0 nL, 2.0 nL, 3.0 nL, 4.0 nL, 5.0 nL, 10.0 nL,20 nL, 30 nL, 40 nL, 50 nL, 60 nL, 70 nL, 80 nL, 90 nL, 100 nL, or morethan 100 nL. In some instances, the calibrated volume comprises at leastor about 100 nL, 200 nL, 300 nL, 400 nL, 500 nL, 600 nL, 700 nL, 800 nL,900 nL, 1000 nL, 2000 nL, 3000 nL, 4000 nL, 5000 nL, 6000 nL, 7000 nL,8000 nL, 9000 nL, 10000 nL, 20000 nL, 30000 nL, 40000 nL, 50000 nL,60000 nL, or more than 60000 nL.

In some instances, the injector introduces one or more dispersed phasesto a microfluidic channel or tube for subsequent reactions in a reactor.In some instances, the one or more dispersed phase is rejected to awaste stream not in fluid communication with the microfluidic channel ortube for subsequent reactions in a reactor. In some instances, theinjector introduces one or more continuous phases. In some instances,the one or more continuous phases comprise oil.

Further embodiments of injectors useful in the systems and methodsprovided herein are described in PCT Publication No. WO2018098438,incorporated herein by reference.

IV. Process System

Systems and methods provided herein can include an injector, alsoreferred to herein as an injection system. In certain embodiments, aninjector serves as an interface between the intake system, also referredto herein as a sampler, autosampler, or similar wording, and the processsystem, where the injector can cycle between a configuration that is influid communication with the intake system and a configuration that isfluid communication with the process system. The injector can beconfigured to have additional configurations, e.g., configurations thatallow cleaning of one or more parts of the intake system and injector.In certain embodiments, the injector comprises common conduit, alsoreferred to herein as an injection chamber, or injection loop, where thecommon conduit can be in fluid communication with the intake system orin fluid communication with the process system, but cannotsimultaneously be in fluid communication with both. Thus, the intakesystem and/or the injector can be treated between injections ofdispersed phase, e.g., between samples, in order to reduce or eliminatetraces of dispersed phase, e.g., sample, between rounds of intake ofdispersed phase, e.g., sample. It will be appreciated that “intakesystem” can include the injector when the system is configured to befluidly connected to the injector, as will be clear from context in thefollowing description.

In certain embodiments, systems and methods provided herein may comprisean intake system, such as any of the intake systems described above,connected to an injector in such a way that in a first configurationthere is fluid communication between the injector and the intake systemand in a second configuration there is not fluid communication betweenthe injector and the intake system. In further embodiments, in thesecond configuration the injector is in fluid communication with aprocess system. The process system can comprise a partitioner, such as apartitioner as described below. The process system can comprise areactor, such a reactor as described below. The process system cancomprise a detector, such as a detector as described below. The processsystem can comprise a disengager, such as a disengager as describedbelow. In certain embodiments, the process system comprises apartitioner and a reactor. In certain embodiments, the process systemcomprises a partitioner, a reactor, and a detector. In certainembodiments, the process system comprises a partitioner, a reactor, adetector, and a disengager. In certain embodiments, the process systemis a system for performing a digital process in partitions, such as adigital PCR system. In certain embodiments, surfaces of the injectorthat come in contact with a dispersed phase, such as a sample, havehigher affinity for at least a first continuous phase than for thedispersed phase; in certain embodiments, surfaces of the injector thatcome in contact with dispersed phase, e.g., sample, comprisefluoropolymer and the at least first continuous phase comprises afluorinated oil.

A. Partitioner

The systems and methods described herein can provide a partitioner, alsoreferred to as a droplet partitioner or droplet generator herein, fordividing dispersed phase into partitions, also referred to as dropletsherein.

In certain embodiments, systems and methods comprise use of an intakesystem and a process system that comprises a partitioner, such as apartitioner as described herein. The intake system can be a system thatcan be completely isolated from the process system, such as an intakesystem as described above, for example, an intake system that comprisesa intake system and, in certain configurations, an injector, where theinjector can alternate between fluid connection with the intake systemand fluid connection with the process system, e.g., where the injectorat no time provides a continuous fluid connection between the intakesystem and the process system. Such intake systems are described morecompletely elsewhere herein. The partitioner may be any suitablepartitioner, e.g., a partitioner that is configured to partition adispersed phase, such as a dispersed phase supplied by an injector (insome cases, packets of dispersed phase surrounded by a continuous phaseand/or separated from each other by a spacer fluid), into a plurality ofpartitions flowing in emulsion in a continuous phase, which can be anysuitable number of partitions, for example as described below, such as1000-100,000 partitions, where the partitions are an average of anysuitable volume, for example as described below, such as 0.05 nL to 5nL. In certain embodiments, the partitioner is a reverse-y partitioner,e.g., a partitioner in which an outlet is positioned within 30 degreesof parallel with a gravitational field and flow through the outlet iscounter to gravitational force, such as those described herein. Incertain embodiments, the partitioner is one in which an inlet fordispersed phase and an inlet for continuous phase meet at an angle of170-190 degrees, such as an angle of 180 degrees (e.g., coaxial).

In certain embodiments, the surfaces of the partitioner that come incontact with dispersed phase (e.g., sample coming from the injector, andpartitions of dispersed phase flowing out of partitioner), and/or withall fluid, have greater affinity for continuous phase than for dispersedphase. Thus, the continuous phase preferentially wets the wall of thechannel, and, for example, preventing wall effects on droplet formationor portions of the dispersed phase being held up in the channel. Incertain embodiments, the surface of the channel (e.g., inlet channel forsample in dispersed phase, outlet channel for partitions of dispersedphase in continuous phase) comprises a fluoropolymer, the continuousphase comprises a fluorinated or perfluorinated oil, and the dispersedphase comprises water or water-based mixture. In certain embodiments,the partitioner can be constructed from one or more blocks of materialthat has a greater affinity for continuous phase than for dispersedphase, for example, one or more blocks of fluoropolymer when thecontinuous phase is, e.g., a fluorinated oil; the block can includeconnections for conduits to channels within the block, where one or moreof the connections minimize or eliminate disruption of flow through theconnection. In certain embodiments, the partitioner includes a firstinlet channel and a second inlet channel, where the first and secondinlet channels intersect at an angle 150-180 degrees, for example at anangle of 170-180 degrees, or at an angle of 180 degrees. In some cases,the inlet channels are oriented so that the direction of flow within thechannels is within 45, or within 30, or within 20, or within 15 degreesof orthogonal to a gravitational field. In certain embodiments, thepartitioner include an outlet channel at the intersection of inletchannels, where the outlet channel is oriented parallel or nearlyparallel to an ambient gravitational field, e.g., within 20, 15, 10, or5 degrees of parallel; in some cases, the direction of flow through theoutlet channel is opposite to the direction of the gravitational field.In certain embodiments, after the partitioner produces an emulsion ofdispersed phase in continuous phase, a portion of the continuous phaseis removed from the emulsion; part or all of the removed continuousphase may be added back to the system, for example, prior to dropletsflowing through a detector in order to separate the droplets beforedetection.

Partitioners described herein may be incorporated into systems forchemical, physical, or biological processing of the dispersed phasecontents. In certain embodiments using injectors as described herein,dispersed phase is not fed to the partitioner continuously, but inpackets already entrained/dispersed in a continuous phase, e.g., packetsthat each represent an individual sample that has been sampled by asampling system; the packets may be separated by a spacer fluid, asdescribed herein. Packets of dispersed phase may be any suitable volume,such as 0.1-100 uL, or 1-80 uL, or 5-50 uL, or 10-30 uL, or 15-25 uL, orany other volume as described herein. If a spacer fluid is used betweenpackets, it may also be any suitable volume, such as 0.1-50 uL, or0.1-20 uL, or 0.1-10 uL, or 1-10 uL, or 1-8 uL, or 2-7 uL, or 3-7 uL, orany other volume as described herein. Thus, dispersed phase may besupplied to the partitioner discontinuously, e.g., as a series ofdiscrete packets, each of which corresponds to, for example, a discretesample, and partitions (droplets) are generated discontinuously. This isa natural consequence of using certain embodiments of injector. As such,the flow will preferably be laminar to avoid breakup of that packet ofdispersed phase or polydispersity of the produced droplets. In addition,in certain embodiments, the droplet partitioner is not greatly affectedby variations in supply flow that may be a consequence of movinginjector positions, which can allow, for example, the loading of a newsample or purge fluid into the continuous stream during dropletformation. Thus, at certain times in the injection cycle, flow from theinjector to the partitioner may be interrupted. Further, in certainembodiments the partitioner is fabricated completely out of materialwith a higher affinity for the continuous phase than for the dispersedphase; for example, in certain embodiments, the partitioner can befabricated out of fluoropolymer, for use with, e.g., a continuous phasecomprising fluorinated oil.

Partitioners of the systems and methods described herein can bereusable, and can partition multiple packets of dispersed phase, e.g.,during a process run, such as packets which each represent a differentsample, in series, such as a series of at least 10, 20, 50, 100, 200,300, 500, or 1000 packets and/or not more than 20, 50, 100, 200, 300,500, 1000, or 10,000 packets.

Thus, described herein are systems for serial flow emulsion reaction,wherein a droplet generator (partitioner) generates droplets(partitions) for reactions. Methods for droplet (partition) generationinclude, but are not limited to, orifices, t-junctions, flow-focusingjunctions, and v-junctions. In some instances, the v-junction is areverse v-junction (also referred to herein as a reverse-y junction) inwhich, in certain embodiments, the channel cross-section restricts afterthe junction rather than before it. In some instances, the dropletgenerator comprises a fluoropolymer. In some instances, the v-junctiondoes not comprise a restriction. In some instances, the droplets aregenerated where two channels meet at an angle less than about 90degrees. In some instances, the angle is at least or about 10 degrees,15 degrees, 20 degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees,45 degrees, 50 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees,75 degrees, or 80 degrees. In some instances, the combined channel goesthrough a reduction in channel diameter. The channel diameter may bereduced by at least 0.5×, 1.0×, 1.5×, 2.0×, 2.5×, 3.0×, 3.5×, 4.0×,4.5×, 5.0×, or more than 5× as compared to a channel that is not reducedin channel diameter. In some instances, a first channel of the twochannels comprises an emulsion of the dispersed phase in a firstcontinuous phase. In some instances, a second channel of the twochannels comprises a continuous phase. In some instances, an exitchannel is orthogonal to the original channel and is orientedvertically. In some instances, the force that moves generated dropletsout of the junction comprises buoyancy effects. In some instances, adroplet size is unaffected by the relative flowrates of the emulsion andthe second continuous phase. In some instances, a droplet size isdetermined by the cross-sectional area of the exit channel.

Thus, in certain embodiments V-junctions may be used for in systems andmethods as described herein. In some instances, the v-junction is areverse v-junction, also referred to herein as a reverse-y. In someinstances, the v-junction comprises two channels. In some instances, across section of each of the two channels is the same. In someinstances, the length of each of the two channels is the same. In someinstances, the two flow channels combine to a single channel. In someinstances, the single channel comprises a reduced cross section ascompared to each of the two channels. In some instances, thecross-section of the single channel is at least or about 0.5×, 1.0×,1.5×, 2.0×, 2.5×, 3.0×, 3.5×, 4.0×, 4.5×, 5.0×, 5.5×, 6.0×, 6.5×, 7.0×,7.5×, 8.0×, 8.5×, 9.0×, 9.5×, or 10× reduced as compared to each of thetwo channels. In some instances, the cross section of the single channelis reduced to a cross section of a droplet. In some instances, thedroplet (e.g., average droplet size) is at least or about 5 micron (um),10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um,120 um, 140 um, 160 um, 180 um, 200 um, or more than 200 um in diameter,and or not more than 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80um, 90 um, 100 um, 120 um, 140 um, 160 um, 180 um, 200 um, 300, 500, or1000 um in diameter, such as 1-1000 um, or 10-500 um, or 50-500 um, or100-400 um, or 100-300 um, or 150-250 um in diameter. In some instances,the droplet is at least or about 200 um, 300 um, 400 um, 500 um, 600 um,700 um, 800 um, 900 um, 1000 um, or more than 1000 um in diameter.

In some instances, the droplet separation and an optical stage arecombined. The droplet separation and optical stage may be on the samemicrofluidic chip. The emulsion stream coming from the thermal cyclermay enter a first channel on the chip. A stream of continuous phase mayenter a second channel on the chip. In some instances, the two channelsboth constrict and then meet at a t-junction. The combined streams flowthrough an extended region at the same constricted size, a portion ofwhich may comprise the optical stage. The constricted channel may thenexpand to a larger diameter and leaves the chip through a press-fitconnection. In some instances, the first channel and the second channeldo not constrict before meeting in a t-junction. In some instances, aconstriction occurs beyond the exit of the t-junction. In someinstances, the constriction is smaller than the characteristic size ofthe droplets so that a droplet substantially fills the channel in theoptical stage. A portion of the constricted channel may form the opticalstage. In some instances, the channel then expands to a larger size sothat it substantially matches the cross-section of an exit tube. In someinstances, the junction is a “v-junction.” In some instances, thestreams are combined at an angle substantially less than 90 degrees, forexamples, at least or about 10 degrees, 15 degrees, 20 degrees, 25degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, 50 degrees, 55degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees, or 80 degrees.

In some instances, a reverse-v junction is used with a tubular opticalstage. In some instances, an exit of the reverse-v junction is tubularand continues to form an optical stage of the detector.

Droplets may be partitioned using a droplet technique. In someinstances, the droplet technique is passive or active. For example,passive techniques include passive flow and passive pressure. Exemplaryactive droplet techniques include, but are not limited to, techniquesbased on mechanical principles (e.g., external syringe pumps, pneumaticmembrane pumps, vibrating membrane pumps, vacuum devices, centrifugalforces, piezoelectric/ultrasonic pumps and acoustic forces); electricalor magnetic principles (e.g., electroosmotic flow, electrokinetic pumps,ferrofluidic plugs, electrohydrodynamic pumps, attraction or repulsionusing magnetic forces and magnetohydrodynamic pumps); thermodynamicprinciples (e.g., gas bubble generation/phase-change-induced volumeexpansion); other kinds of surface-wetting principles (e.g.,electrowetting, and optoelectrowetting, chemically, thermally,structurally and radioactively induced surface-tension gradients);gravity; surface tension (e.g., capillary action); electrostatic forces(e.g., electroosmotic flow); centrifugal flow (substrate disposed on acompact disc and rotated); magnetic forces (e.g., oscillating ionscauses flow); magnetohydrodynamic forces; and vacuum or pressuredifferential.

The droplet generator may generate droplets comprising various volumes.In some instances, a volume of the droplet comprises at least or about0.01 nanoliter (nL), 0.02 nL, 0.03 nL, 0.04 nL, 0.05 nL, 0.06 nL, 0.07nL, 0.08 nL, 0.09 nL, 0.10 nL, 0.20 nL, 0.30 nL, 0.40 nL, 0.45 nL, 0.50nL, 0.55 nL, 0.60 nL, 0.65 nL, 0.70 nL, 0.75 nL, 0.80 nL, 0.90 nL, 1.0nL, 2.0 nL, 3.0 nL, 4.0 nL, 5.0 nL, 10.0 nL, 20 nL, 30 nL, 40 nL, 50 nL,60 nL, 70 nL, 80 nL, 90 nL, 100 nL, or more than 100 nL and/or not morethan 0.02 nL, 0.03 nL, 0.04 nL, 0.05 nL, 0.06 nL, 0.07 nL, 0.08 nL, 0.09nL, 0.10 nL, 0.20 nL, 0.30 nL, 0.40 nL, 0.45 nL, 0.50 nL, 0.55 nL, 0.60nL, 0.65 nL, 0.70 nL, 0.75 nL, 0.80 nL, 0.90 nL, 1.0 nL, 2.0 nL, 3.0 nL,4.0 nL, 5.0 nL, 10.0 nL, 20 nL, 30 nL, 40 nL, 50 nL, 60 nL, 70 nL, 80nL, 90 nL, 100 nL, or 200 nL. In some instances, a volume of the dropletcomprises no more than 0.75 nL. In some instances, a volume of thedroplet comprises about 0.01 nL to about 100 nL, about 0.05 nL to about80 nL, 0.05 nL to about 50 nL, 0.05 nL to 25 nL, 0.05 nL to 10 nL, 0.05nL to 5 nL, about 0.10 nL to about 60 nL, about 0.10 nL to about 40 nL,about 0.10 nL to about 20 nL, about 0.10 nL to about 10 nL, about 0.10nL to about 5 nL, about 0.10 nL to about 1 nL, about 0.2 nL to about 40nL, about 0.2 nL to about 20 nL, about 0.2 nL to about 10 nL, about 0.2nL to about 1 nL about 0.2 nL to about 0.8 nL, about 0.3 nL to about 30nL, 0.3 nL to about 10 nL, 0.3 nL to about 1 nL, 0.3 nL to about 0.7 nL,about 0.4 nL to about 20 nL, 0.4 nL to about 10 nL, 0.4 nL to about 1nL, 0.4 nL to about 0.6 nL, about 0.5 nL to about 10 nL, about 0.6 nL toabout 4 nL, or 0.7 nL to about 2.0 nL. In certain embodiments, averagedroplet volume is 0.1-1 nL.

In some instances in which droplets are formed from sample containingnucleic acid, the sample is partitioned into droplets having, onaverage, 0.001 to 200, 0.1 to 2, 0.5 to 2.0, 0.1 to 20, 0.5 to 1.3, or0.1 to 1 nucleic acid molecules. In some cases, one or more droplets donot comprise a nucleic acid molecule. In some instances, one or moredroplets comprise a single nucleic acid molecule. In some cases, one ormore droplets comprise two or more nucleic acid molecule. In someinstances, the nucleic acid molecule is DNA. In some instances, thenucleic acid molecule is RNA. In some instances, the nucleic acidmolecule is single stranded or double stranded.

In some instances, a number of droplets generated by any of the methodsdescribed herein, e.g., from a single sample (packet, as describedabove), is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 4000, 6000, 8000, 10000, 12000, 14000, 16000, 18000, 20000,25000, 30000, 40000, 50000, 100000, 500000, 1000000, or more than1000000 and or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 4000, 6000, 8000, 10000, 12000, 14000, 16000, 18000,20000, 25000, 30000, 40000, 50000, 100000, 500000, 1000000, or 10000000droplets, for example, 100-1,000,000, or 500-500,000, or 1000-100,000,or 2000-50,000, or 10,000-50,000, or 15,000-30,000, or 15,000-25,000droplets. In some cases, the number of droplets is about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 4000, 6000, 8000,10000, 12000, 14000, 16000, 18000, 20000, 25000, 30000, 40000, 50000,100000, 500000, 1000000, or more than 1000000 droplets. In someinstances, at least or about 10, 20, 30, 40, 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 2000, 4000, 6000, 8000, 10000, or more than 10000 droplets aregenerated per second. In some instances, a range of about 10-10000,20-8000, 30-7000, 40-6000, 50-5000, 60-4000, 70-3000, 80-2000, 100-1000droplets are generated per second. In some instances, droplets aregenerated at rate of at least or about 500 ul, 750 ul, 1 mL, 1.5 mL, 2mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, 5 mL, 5.5 mL, 6 mL, 6.5 mL, 7mL, 7.5 mL, 8 mL, or more than 8 mL per hour.

A partitioner typically includes a first inlet channel for dispersedphase (e.g., a programmed emulsion of dispersed phase in continuousphase, supplied by an intake system, such as by an injector), one ormore second inlet channels for continuous phase, and an outlet channelthrough which an emulsion of droplets formed by the partitioner in thecontinuous pass to the rest of the system. Any suitable arrangement ofinlets and outlet may be used. In some cases, the surfaces of thepartitioner, e.g., inlet and outlet channels, that come into contactwith dispersed phase have greater affinity for continuous phase than fordispersed phase, for example, a fluoropolymer if the continuous phasecomprises a fluorinated oil. The channels may be formed in a solid blockof suitable material, such as a material that has a greater affinity forcontinuous phase than for dispersed phase, and in these and other cases,all channels in the partitioner can have greater affinity for continuousphase than dispersed phase; additionally or alternatively, part or allof one or more channels may be formed by tubing inserted into channelsin a block. In this case, the tubing carrying dispersed phase (e.g.,dispersed phase inlet and outlet tubing) can have greater affinity forcontinuous phase than for dispersed phase. Tubing or channels carryingonly continuous phase, e.g., a continuous phase inlet, does notnecessarily have to have greater affinity for continuous phase thandispersed phase (though, for convenience of construction or otherreasons it may), so long as it is constructed of material compatiblewith the continuous phase.

The dispersed phase can contain any suitable material, e.g., in certainembodiments the dispersed phase comprises biological sample, such as asample comprising nucleic acids, for example a sample in a packet in anysuitable volume range, such as any of the volume ranges describedherein. In certain embodiments the dispersed phase may further comprisereagents for the amplification of nucleic acids, detection, and/or othersuitable reagents.

Any suitable partitioner may be used in systems and methods describedherein. Exemplary partitioners include, but are not limited to,reverse-y (also referred to as reverse-v) partitioners, T-junctionpartitioners, and flow-focusing partitioners, described below.

Reverse-y partitioners. In certain embodiments, the partitioner may beconfigured as a “reverse-Y”, also referred to as a “v” or “reverse-v”herein. The reverse-y partitioner is advantageous in that it can producepartitions of the same or substantially the same size over a wide rangeof inlet flow rates. In certain embodiments, surfaces of the reverse-ypartitioner that come in contact with dispersed phase (e.g.,sample/dispersed phase inlet, and outlet), have greater affinity forcontinuous phase than for dispersed phase; in certain embodiments, allsurfaces of the partitioner have greater affinity for continuous phasethan for dispersed phase.

In certain embodiments, described herein are reaction systems andmethods, such as a digital PCR system and methods, in which a reverse-ypartitioner is used to partition sample into a plurality of partitions,which are reacted, e.g. thermally cycled, and detected. In these systemsand methods, any suitable reverse-y partition generator, as describedfurther herein, may be used.

The reverse-y droplet partitioner includes an outlet channel parallel toor close to parallel to the gravitational field and two inlet channelsplaced relatively orthogonal to the gravitational field. See, e.g.,FIGS. 38, 39A and 39B, and 40. In the reverse-y partitioner, two inletchannels meet at an angle (theta) between 2 degrees and 180 degreesbetween the axes of the channels, for example, between 10 and 180degrees, or between 30 and 180 degrees, or between 50 and 180 degrees,or between 90 and 180 degrees, or between 110 and 180 degrees, orbetween 130 and 180 degrees, or between 150 and 180 degrees, or between160 and 180 degrees, or between 170 and 180 degrees between the two axesof the channels, for example, 180 degrees (e.g., coaxial orsubstantially coaxial). See, e.g., FIG. 40 for an embodiment where theinlet channels are co-axial. The inlet channels may be oriented above orbelow the plane orthogonal to the outlet channel (e.g., +/−60, 50, 45,40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 degrees) (alpha). The firstand the second inlet channel can be oriented with a substantialcomponent (e.g., axis of flow) orthogonal to the ambient gravitationalfield, for example, the first inlet channel and/or the second inletchannel can be within 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1degrees of orthogonal to the ambient gravitational field. See, e.g.,FIGS. 38 and 39A and 39B, which shows exemplary arrangements of thefirst and second inlet channels. In the first inlet channel, a mixtureof a first continuous phase and dispersed phases is flowing, with thecontinuous and dispersed phases being mostly immiscible. This is thecase, e.g., when a series of samples is injected from the injector; eachsample is dispersed phase, surrounded or substantially surrounded bycontinuous phase, for example, an aqueous phase surrounded by an oil,e.g., a fluorinated oil. Thus, this continuous and disperse phases cancomprise a programmed emulsion containing discrete sample plugs(packets) separated by continuous phase and/or a spacer fluid. In thesecond inlet channel, fluid miscible with the first continuous phase isflowed; e.g., a second continuous phase. Any suitable fluid may be used.In certain embodiments, the first continuous phase is of the samecomposition as the second continuous phase, e.g., an oil, such as afluorinated oil. In some cases, the first and second continuous phasescomprise an oil and surfactant, such as a surfactant of a type andconcentration described herein. In some cases, the dispersed phasecomprises an aqueous solution. The first and second continuous phase canbe composed of the same oil with varying surfactant concentrations.Additionally or alternatively, surfactant composition and/orconcentration in the first continuous phase can be different fromsurfactant in the second continuous phase. The outlet channel receivesflow from the first inlet channel and the second inlet channel at thejunction point of the first inlet channel and the second inlet channel,and this outlet channel can be oriented parallel or nearly parallel tothe ambient gravitational field, for example, within 45, 40, 30, 35, 20,15, 10, 5, 4, 3, 2, or 1 degree of parallel to the ambient gravitationalfield, such as within 30 degrees, e.g., within 15 degrees, or within 10,5, or 2 degrees. Fluid flow in the outlet channel moves counter to thegravitational force.

Without being bound by theory, it is thought that in orienting thechannels in such a way, buoyancy can play a role in droplet formation.It is possible that buoyancy accelerates the continuous phase resultingin a neck formed behind the droplet. As the neck grows thin enough, iteventually breaks due to Rayleigh-Plateau instability. Thus, orientationof the outlet channel may be important to ensure droplet formation asopposed to separated, two phase flow (as typically seen in Y-junctionsin microfluidic systems). If the outlet channel is oriented orthogonallyor substantially orthogonally to gravity (e.g., within 30 degrees of thedirection of gravity) and fluidic flow is in the same direction asgravitational force, the heavier phase may settle to the bottom of theoutlet channel and the lighter phase may flow above it, generating twophase flow rather than partitioning of the inlet dispersed phase intodroplets. For example, if the droplet partitioner outlet is parallel togravity, the partitioner may not produce monodisperse droplets butrather producer large polydisperse droplets.

The characteristic dimension of the first inlet channel may be largerthan the characteristic dimension of the second inlet channel, viceversa, or the two characteristic dimensions may be similar or equal. The“characteristic dimension,” (also referred to herein as “criticaldimension,” “characteristic length,” and equivalent terms) as that termis used herein, includes any dimension that substantially determines thecross-sectional area of the channel; for example, a characteristicdimension can be a diameter, a hydraulic diameter, or any other suitabledimension. The first and/or second inlet channel can thus have anysuitable characteristic dimension. In certain embodiments, thecharacteristic dimension of the first and/or second inlet channel, e.g.,diameter if the channel has a circular cross-section, can be at least 1,10, 20, 50, 70, 80, 90, 100, 110, 120, 130, 150, 170, 200, 250, 300,400, 500, 600, 700, 800, or 900 um, and/or not more than 10, 50, 70, 80,90, 100, 110, 120, 130, 150, 170, 200, 250, 300, 400, 500, 600, 700,800, 900, or 1000 um, for example, 1-1000 um, or 10-800 um, or 10-600um, or 20-400 um, or 50-300 um, or 100-300 um, or 150-250 um.

The characteristic dimension of the outlet channel can be smaller thanor equal to the characteristic dimensions of the two inlet channels, andit is this dimension that controls the resulting equivalent sphericaldiameter of the droplets that are formed. The characteristic dimensionof the outlet channel, e.g., diameter if the channel has a circularcross-section, can be at least 1, 10, 20, 50, 70, 80, 90, 100, 110, 120,130, 150, 170, 200, 250, 300, 400, 500, 600, 700, 800, or 900 um, and/ornot more than 10, 50, 70, 80, 90, 100, 110, 120, 130, 150, 170, 200,250, 300, 400, 500, 600, 700, 800, 900, or 1000 um, for example, 1-1000um, or 10-800 um, or 10-600 um, or 20-400 um, or 20-300 um, or 20-200um, or 50-200 um, or 80-150 um, or 80-120 um, or 90-110 um, or about 70,80, 90, 100, 110, 120, 130, or 140 um. Unless otherwise noted,dimensions for conduits are interior dimensions. Also unless otherwisenoted, conduits may have any suitable cross-section, such as square,oblong, circular, and the like; in certain cases cross-section may bedescribed as circular but it is to be understood that any suitablecross-section may be used. The outlet channel may or may not expand to alarger characteristic dimension after droplet formation. However, theoutlet channel of the droplet partitioner should be a uniformcharacteristic dimension (e.g., diameter) for a length at least 1.25×that characteristic dimension (e.g., diameter), for example, at least1.25, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times the characteristic(dimension, e.g., diameter, and/or not more than 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, or 15 times the characteristic dimension, e.g., diameter,for example, between 1.5× and 10×, or between 2× and 8×, such as between3× and 7×. For example, if the outlet channel is circular with adiameter of 100 um, typically it should remain at 100 um diameter for alength of at least 125 um. Beyond this region, the outlet conduit,either within the partitioner (e.g., a block of material) or outside thepartitioner (e.g., block), can change to a different characteristicdimension (e.g., diameter) e.g., expand to a greater characteristicdimension (e.g., diameter). In addition, in some embodiments, at somepoint after the outlet conduit its orientation will become perpendicularor nearly perpendicular to the gravitational field, e.g., when leadingto a reactor and/or detector. This can help reduce buoyancy effects onpartitions, which can lead to undesirable axial dispersion of partitionsor other problems.

An advantage of this approach is that the droplet size is relativelyinsensitive to the relative flow rates of fluid in the first inletchannel and the second inlet channel, for example, over a range of atleast 2-fold, or a range of at least 5-fold, or a range of at least10-fold, or a range of at least 100-fold, or a range of at least1000-fold, for example, in flow rates from 0.01-3000 uL/min. Meandroplet size can vary over these ranges by, e.g., not more than 100, 90,80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5, 2, or 1%.

In certain embodiments, the partitioner, e.g., a reverse-y partitioner,is electrically grounded.

FIGS. 38 and 39A and 39B show a reverse-y partitioner. The reverse-ydroplet partitioner comprises an outlet channel 3801, 3901 substantiallyparallel and two inlet channels placed relatively orthogonal to thegravitational field. In the reverse-y partitioner, two small inletchannels 3802, 3902 and 3803, 3903 meet at an angle (theta) between 2degrees and 180 degrees between the axes of the channels. The inletchannels may be oriented above or below the plane orthogonal to theoutlet channel (+−45 degrees) (alpha). The first 3802, 3902 and thesecond 3803, 3903 inlet channels are oriented with a substantialcomponent orthogonal to the ambient gravitational field. The channelsmay be formed in a substrate 3804, 3904. In a first inlet channel 3802,3902, a mixture of continuous and dispersed phases is flowing, with thecontinuous and dispersed phases being mostly immiscible. This continuousand disperse phased comprises a programmed emulsion containing discretesample plugs separated by continuous phase. In a second inlet channel3803, 3903, fluid substantially miscible with the continuous phase isflowed. In certain embodiments, the first continuous phase is of thesame composition as the second continuous phase. In certain embodiments,the first and second continuous phases comprise an oil and surfactant.In certain embodiments, the dispersed phase comprises an aqueoussolution. In certain embodiments, the first and second phase arecomposed of the same oil with varying surfactant concentrations. Incertain embodiments, the surfactant in the first continuous phase isdifferent from the surfactant in the second continuous phase. An outletchannel 3801, 3901 receives flow from the first inlet channel 3802, 3902and the second inlet channel 3803, 3903 at the junction point 3805, 3905of the first inlet channel 3802, 3902 and the second inlet channel 3803,3903, and this outlet channel 3801, 3901 is oriented substantiallyparallel to the ambient gravitational field. Fluid flow in the outletchannel 3801, 3901 moves counter to the gravitational force. Inorienting the channels in such a way, buoyancy can play a role indroplet formation. The outlet channel 3801, 3901 may or may not expandto a larger length after droplet formation (25 um-2000 uM). Thecharacteristic length of the first inlet channel 3801, 3901 may belarger than the characteristic length of the second inlet channel 3802,3902, vice versa, or the two characteristic lengths may be substantiallysimilar. The characteristic length can be a diameter, a hydraulicdiameter, or any length scale that substantially determines thecross-sectional area of the channel. (1-1000 um) The characteristiclength of the outlet channel 3801, 3901 is smaller than or equal to thecharacteristic lengths of the two inlet channels, and it is this lengththat controls the resulting diameter of the droplets that are formed.(1-1000 um) An advantage of this approach is that the droplet size isrelatively insensitive to the relative flow rates of fluid in the firstinlet channel and the second inlet channel. (0.01-3000 uL/min).

FIGS. 44A and 44B and 45 illustrate embodiments of a reverse-ypartitioner in which the two inlet channels intersect at 180 degrees,are orthogonal to the gravitational field, and intersect with the outletchannel at an angle of 90 degrees, with the outlet channel orientedparallel to the gravitational field. In this embodiment, fluid flow ofthe first inlet channel collides with fluid flow of the second inletchannel. In certain embodiments, including that illustrated in FIGS.44A, 44B, and 45, one, two, or all three of the channels are containedwithin a solid block. The channels of the partitioner, of any form, areconnected to the rest of the system. FIGS. 44A and 44B and 45 alsoillustrate three ways in which tubing to connect the partitioner to therest of the system may be used. FIG. 45 illustrates an embodiment inwhich tubing is attached with specialized adapters, where the channelsof the partitioner are fluidic channels in the block. FIG. 44Aillustrates an embodiment in which tubing is inserted partway into theblock, where the channels are tubing channels connected to fluidicchannels; in the case of FIG. 44B, tubing is inserted all the way intothe block, thus the channels are tubing channels. Any combination of thethree methods may be used. Thus, channels that come in contact withfluids may be formed by drilling or milling or similar operation, bytubing, or any combination thereof. Connections are described in moredetail below. In instances in which tubing is inserted all the way intothe block, the tubing itself serves as the channel (inlet and/oroutlet). In all cases, in certain embodiments, surfaces that come incontact with fluids, such as with dispersed phase, have a higheraffinity for continuous phase than for dispersed phase.

Thus in certain embodiments the channel geometries may be as describedabove. In certain embodiments, the first and second inlet channels areoriented 180 degrees apart, so that a single drilling or milling pathwayis used to produce these channels. The outlet channel is created by adrilling or a milling operation that connects with the inlets channels.Each inlet or outlet channel may be comprised of a single drilledfluidic channel, a single drilled tubing channel, or a combination ofthe two. (A) Fluidic channels. (B) Tubing channels connected to fluidicchannels. (C) Tubing channels.

In a first embodiment, e.g. as shown in FIG. 45, channels are generatedwith inner diameters equal to the characteristic length for dropletformation. The channels are generated by two or more drillingoperations. The first of said drilling operations generates the twoinlets channels 4502 and 4503. The second of said drilling operationsgenerates the outlet 4501. The second drilling operation connects thesecond channel to the first channel generated by the first drillingoperation forming a junction 4512. Tubing 4505 may be attached to thedroplet partitioner in any suitable, manner, such as by a compressionfitting using a nut and ferrule. The fitting generates a fluidic sealbetween the partitioner and tubing as the fitting is tightened.

In a second embodiment, e.g., as shown in FIG. 44A, channels aregenerated with two distinct inner diameters. The first inner diameter isequal to the characteristic length for droplet formation. The secondinner diameter is of a size to connect tubing through an interferencefit. The drilled channels comprise an outlet with a first channel 4401with an inner diameter equal to the characteristic length for dropletformation and a second channel 4406 with an inner diameter equal to orsmaller than the OD of the tubing 4405 to be inserted. When insertingtubing 4405 with a higher elastic modulus than the block material, asufficient force is applied to the tubing forming a fluidic seal. Thedrilled channels also comprise two inlets with distinct inner diameters.For the first inlet, the drilled channels comprise a first channel 4402with an inner diameter of a size capable of transporting the first fluidand a second channel 4407 with an inner diameter equal to or smallerthan the OD of the tubing 4405 to be inserted. For the second inlet, thedrilled channels comprise a first channel 4403 with an inner diameter ofa size capable of transporting the first fluid and a second channel 4408with an inner diameter equal to or smaller than the OD of the tubing4405 to be inserted. The first 4402 and second 4403 inlet channels maybe of equal or different sizes. The first inner diameter of the outletchannel 4401 is equal to the characteristic length for partitionformation. The channels may be generated using one or more drillingoperations that form a junction 4412 capable of partition formation. Ina third embodiment, such as shown in FIG. 44B, channels are generatedwith a single distinct inner diameter equal to or smaller than the OD ofthe tubing 4405. The channels are generated by two or more drillingoperations. The first of said drilling operations generates the twoinlets channels 4410 and 4411. The second of said drilling operationsgenerates the outlet 4409. The second drilling operation connects thesecond channel to the first channel generated by the first drillingoperation forming a junction. When inserting tubing 4405 with a higherelastic modulus than the block material, a sufficient force is appliedto the tubing forming a fluidic seal. Insertion of the tubing into thevery center of the substrate 4404 forms a junction 4413. The innerdiameter of the tubing is used as the channels and the critical diameterfor partition formation is determined by the critical diameter of theinner diameter of the outlet tubing.

In certain embodiments, any combination of tubing connection methods maybe used.

The droplet partitioner channels in the block, e.g., fluoropolymer blockare made sufficiently small to ensure stable fluid flow reducing thepossibility for hold up of sample in the channels.

In a preferred embodiment the tubing ID and channel ID are size matchedto ensure stable fluid flow reducing the possibility for hold up ofsample in the channels.

Droplet size for reverse-y partitioner. Methods for controlling the sizeof droplets produced in the droplet partitioner comprise controlling thecross-sectional area of the outlet channel, for example, controlling acharacteristic dimension, also referred to herein as “characteristiclength,” or “critical dimension,” such as a diameter or a hydraulicdiameter. Droplets will be similar in diameter to this characteristic(critical) dimension, and/or have average cross-sectional areas similarto the cross-sectional area of the outlet channel. This differs fromorifice-style droplet partitioners, where droplets that are formed willgenerally be substantially larger than the characteristic dimension ofthe orifice, generally larger than one and half times the characteristicdimension. An advantage of this style of droplet partitioner is that thedroplet size is relatively insensitive to variability of the flowratefrom the first inlet channel and the second inlet channel, as thecharacteristic dimension of the outlet channel generally controls thediameter of the produced droplets. This differs from shear-based (e.g.T-junction or cross-junction) or orifice droplet partitioner, whichgenerally require stable flowrates to produce monodisperse droplets. Assuch, a system employing this droplet partitioner may be able to useless expensive, complicated, or bulky means of supplying the necessarydriving force to introduce fluids into the first inlet channel and thesecond inlet channel. When referring to droplet diameter, it is meantthe characteristic or equivalent spherical diameter (the diameter thatthe droplets would take as spheres suspended in the continuous phase).Droplets constrained within channels may elongate and have a criticaldimension orthogonal to the channel axis shorter than this equivalentspherical diameter. Thus, in some embodiments, the systems and methodsdescribed herein, e.g., digital PCR methods, provide a plurality ofpartitions (droplets), such as at least 100, 1000, or 10,000 partitions,from a single sample (e.g., the dispersed phase that enters thepartitioner in an inlet channel), where the partitions have an meanspherical diameter, such as a mean spherical diameter in the range of10-1000, 20-800, 30-700, 40-500, 40-400, 40-300, 50-200, 50-150, or75-125 um and wherein the coefficient of variation of the sphericaldiameters of the partitions is less than 50, 40, 30, 20, 15, 12, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1%, over a range of flow rates in inlet channelsof at least 10-fold, or at least 100-fold, or at least 1000-fold.

T-junction partitioners. FIGS. 41A, 41B, and 41C show another system forpartitioning droplets (a “T-junction” droplet partitioner). In theT-junction partitioner, two inlet channels meet at an angle (theta)between 2 degrees and 90 degrees between the axes of the channels andconverge to a single outlet channel. In some cases, the inlet channelsmeet at an angle between 90 and 180 degrees, or between 110 and 180degrees, or between 130 and 180 degrees, or between 150 and 180 degrees,or between 160 and 180 degrees, or between 170 and 180 degrees, in somecases 180 degrees (co-axial); an arrangement in which the inlet channelsare co-axial is shown in FIG. 41C. In a first inlet channel, a mixtureof continuous and dispersed phases is flowing, with the continuous anddispersed phases being mostly immiscible. In a second inlet channel,fluid substantially miscible with the continuous phase is flowed.Continuous phases, surfactants, etc., can be as described for thereverse-y partitioner. The first and the second inlet channel can beoriented in any suitable plane relative to the ambient gravitationalfield, such as with a substantial component perpendicular to the ambientgravitational field. An outlet channel receives flow from the firstinlet channel and the second inlet channel at the junction point of thefirst inlet channel and the second inlet channel, and this outletchannel is also oriented in any suitable manner, e.g., perpendicular orsubstantially perpendicular to the ambient gravitational field. In somecases, the flow of sample is perpendicular or nearly perpendicular(e.g., angle of 60-90, 70-90, 80-90, or 85-90 degrees) to the flow ofthe continuous phase (see, e.g., FIG. 41A), with sample phase parallelto the outlet. In some cases, the outlet and continuous phase areparallel and perpendicular or nearly perpendicular (e.g., angle of60-90, 70-90, 80-90, or 85-90 degrees) to the sample phase. This resultsin droplets being pushed off into smaller partitions (see, e.g., FIG.41B). In some cases, the sample phase and the continuous phase areparallel with each other (see, e.g., FIG. 41C), i.e., intersect at 180degrees or nearly 180 degrees, and the outlet is perpendicular or nearlyperpendicular (e.g., angle of 60-90, 70-90, 80-90, or 85-90 degrees) tothe inlets. In this case, the two phases are colliding head-on. Theoutlet channel may or may not expand to a larger length after dropletformation. The characteristic dimension of the first inlet channel maybe larger than the characteristic dimension of the second inlet channel,vice versa, or the two characteristic dimensions may be substantiallysimilar. In certain embodiments, the characteristic dimension of thefirst and/or second inlet channel, e.g., diameter if the channel has acircular cross-section, can be at least 1, 10, 20, 50, 70, 80, 90, 100,110, 120, 130, 150, 170, 200, 250, 300, 400, 500, 600, 700, 800, or 900um, and/or not more than 10, 50, 70, 80, 90, 100, 110, 120, 130, 150,170, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 um, forexample, 1-1000 um, or 10-800 um, or 10-600 um, or 20-400 um, or 50-300um, or 100-300 um, or 150-250 um. The characteristic dimension of theoutlet channel is either smaller than, equal to, or larger than thecharacteristic lengths of the two inlet channels. In certainembodiments, the characteristic dimension of the outlet channel, e.g.,diameter if the channel has a circular cross-section, can be at least 1,10, 20, 50, 70, 80, 90, 100, 110, 120, 130, 150, 170, 200, 250, 300,400, 500, 600, 700, 800, or 900 um, and/or not more than 10, 50, 70, 80,90, 100, 110, 120, 130, 150, 170, 200, 250, 300, 400, 500, 600, 700,800, 900, or 1000 um, for example, 1-1000 um, or 10-800 um, or 10-600um, or 20-400 um, or 50-300 um, or 100-300 um, or 150-250 um. Dropletsize is determined by the relative flow rates and geometry of the twoinlet channels. As with other partitioners described herein, thispartitioner can be fabricated using one or more solid blocks as astarting point, for example, a fluoropolymer block or similar block thathas greater affinity for continuous phase than for dispersed phase, orcan have coated surfaces, or conduits, or other features that conveygreater affinity for continuous phase than for dispersed phase, inparticular, in channels that come in contact with dispersed phase.

Flow-focusing droplet partitioner FIG. 42 shows another system forpartitioning droplets (a “flow focusing” droplet partitioner, alsoreferred to herein as a cross-junction partitioner). Two continuousphase flows result in the pinching of a plug into multiple smallerpartitions. In the flow focusing partitioner, three inlet channels meetat an angle (theta) between 2 degrees and 90 degrees between the axes ofthe channels and converge to a single outlet channel. In some cases, thetwo outer channels are oriented perpendicular to the interior inletchannel, as shown in FIG. 42. In the interior inlet channel, a mixtureof continuous and dispersed phases is flowing, with the continuous anddispersed phases being mostly immiscible. In the external inletchannels, a fluid substantially miscible with the continuous phase isflowed. Continuous phase, surfactants, and the like may be as describedfor the reverse-y droplet generator. All channels can be oriented in anysuitable manner, e.g., with a substantial component perpendicular to theambient gravitational field. An outlet channel receives flow from thethree inlet channels and this outlet channel is also oriented in anysuitable manner, e.g., substantially perpendicular to the ambientgravitational field. The outlet channel may or may not expand to alarger length after droplet formation. The characteristic dimension ofthe interior channel may be larger than the characteristic dimensions ofthe outer inlet channels, vice versa, or the characteristic dimensionsmay be substantially similar. In certain embodiments, the characteristicdimension of one or both of the outer inlet channels and/or the interiorinlet channel, e.g., diameter if the channel has a circularcross-section, can be at least 1, 10, 20, 50, 70, 80, 90, 100, 110, 120,130, 150, 170, 200, 250, 300, 400, 500, 600, 700, 800, or 900 um, and/ornot more than 10, 50, 70, 80, 90, 100, 110, 120, 130, 150, 170, 200,250, 300, 400, 500, 600, 700, 800, 900, or 1000 um, for example, 1-1000um, or 10-800 um, or 10-600 um, or 20-400 um, or 50-300 um, or 100-300um, or 150-250 um. The characteristic length of the outlet channel iseither smaller than, equal to, or larger than the characteristic lengthsof the two inlet channels. In certain embodiments, the characteristicdimension of the outlet channel, e.g., diameter if the channel has acircular cross-section, can be at least 1, 10, 20, 50, 70, 80, 90, 100,110, 120, 130, 150, 170, 200, 250, 300, 400, 500, 600, 700, 800, or 900um, and/or not more than 10, 50, 70, 80, 90, 100, 110, 120, 130, 150,170, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 um, forexample, 1-1000 um, or 10-800 um, or 10-600 um, or 20-400 um, or 50-300um, or 100-300 um, or 150-250 um. Droplet size is determined by therelative flow rates and geometry of the two inlet channels. As withother partitioners described herein, this partitioner can be fabricatedusing one or more solid blocks as a starting point, for example, afluoropolymer block or similar block that has greater affinity forcontinuous phase than for dispersed phase, or can have coated surfaces,or conduits, or other features that convey greater affinity forcontinuous phase than for dispersed phase.

Surfactants In some embodiments, the continuous phase, the dispersedphase, or both comprise a surfactant to stabilize the size of packets ofcontinuous phase in dispersed phase. Surfactant composition andconcentration may be as described elsewhere herein, e.g., see “Materialsused in systems and embodiments provided herein,” above. In certainembodiments, continuous phase flowing in an inlet of the partitioner,such as a fluorinated oil, comprises a surfactant, such as a fluorinatedsurfactant, at a concentration of 0.2-2%, such as 0.5-1.5%, or 0.8-1.2%,or any other concentration as described herein.

Partitioner integration into the overall system The partitioner istypically incorporated into a larger system for conducting chemical,physical, or biological processing. Systems and methods provided hereininclude a partitioner that is enclosed by a holder and/or that isconnected to the overall system as described herein. Supply ofcontinuous phase or mixtures of continuous phase and dispersed phase canbe provided to the inlets of the droplet partitioner using tubes, pipes,microfluidic channels, or any other suitable systems and methods ofsupplying fluids. Mixtures of continuous phase and produced droplets canbe connected to downstream processing elements using tubes, pipes,microfluidic channels, or any other suitable systems and methods ofconveying fluids. In certain embodiments, polymer tubing is used tosupply fluids to at least one of the inlets or convey fluid from theoutlet. In some cases, tubing or other conduits that come in contactwith dispersed phase have a greater affinity for continuous phase thanfor dispersed phase. In some cases, this tubing comprises afluoropolymer.

It will be appreciated that, although connections are described in thissection for connections to a partitioner, connections in other parts ofthe system (e.g., at a droplet separator, a detector, an injector, orany other suitable point) may also use similar or identical systems andmethods, and the descriptions in this section apply to other areas inthe system, as appropriate.

Typically, connections are formed in such a way as to reduce oreliminate dead spaces and other potential disruptions to flow in thesystem. In general, when flow moves in a conduit, connections or othereffects that expand the characteristic dimension, e.g., diameter of theconduit are less likely to create dead spaces than connections or othereffects where diameter remains constant; typically, it is not desirableto have connections where characteristic dimension, e.g., diameterdecreases, especially if it decrease abruptly, as this can lead to deadzones or other disruptions. In certain embodiments, the cross-sectionaloutline of a conduit prior to a connection is matched with thecross-sectional outline of the conduit after the connection by theconnection itself in such a manner that the two cross-sectional outlinesare identical or nearly identical, and there is no or substantially nobreak at the junction between the two conduits. Conduits, e.g., tubingmay be attached to the droplet partitioner in any suitable manner, e.g.,by an interference fit or a compression fitting. See, e.g., FIGS. 43A,43B, and 43C. In the first instance (FIG. 43C) holes are created in theblock that is the droplet partitioner, e.g., drilled into a solid block,such as a block that has greater affinity for continuous phase than fordispersed phase, e.g., a fluoropolymer block for use with, e.g.,fluorinated oils, with equal or slightly smaller diameter as the outerdiameter of the tubing to be inserted. When inserting tubing with ahigher elastic modulus than the block material, a sufficient force isapplied to the tubing forming a fluidic seal. Since the drilled hole issmaller than the tube and the tube is more rigid than the block it formsa fluidically tight seal. In certain embodiments, the cross-sectionaloutline of the tubing is identical or nearly identical to thecross-section outline of the block when the tubing is inserted (e.g.,with circular tubing and circular conduit in the block, the ID of thetubing is the same or nearly the same as the block when the tubing isinserted into the block, e.g., within 5, 2, 1, 0.5, 0.1, 0.05, 0.01,0.005, or 0.001% of the same). The end of the tubing fits snugly againstthe block, so that, at flow rates and with compositions used in thetubing and the block, no disruption in flow or dead spots are created;in certain embodiments, the end of the tubing inserted into the block isconfigured to snug against the block material for the entirecircumference of the tubing. Typically this can be accomplished bycreating smooth cuts in the tubing that are perpendicular to the axis offlow in the tubing, and having a complementary surface for the tubing tobutt against in the block. In the second instance (FIG. 43A), acompression fitting and nut is used to create a fluidic seal between thetubing and the channel in the block, e.g., block as described herein,such as fluoropolymer block. Any suitable combination of fluidic sealsmay be used. FIG. 43 also shows the partitioner block, e.g., block asdescribed herein such as fluoropolymer block, that has been manufacturedwith the channels, contained in a holder, or housing material, toprovide mechanical stability. This housing material may be composed ofany suitable material that provides the desired mechanical stability,and capable of operations to shape the material, including machining,engraving, etching, ablating, embossing, molding, or printing forexample, a metal, such as aluminum or stainless steel, for mechanicalstrength. FIG. 43B tubing is shown that is held in place using fittingsthat provide extra stability so the tubing doesn't get pulled out. FIGS.43B and 43D show the use of specialized fittings. The fittings use a nutand ferrule to hold the tube in place against the polymer block. Theferrule crimps down on the tube while also providing pressure againstthe block. The nut should be able to reach down into the block so thehousing material can't be so large that it does not reach. It ispreferred that the tubing diameter matches the channel diameter toensure stable fluid flow.

Thus, as in FIG. 43, in a first embodiment, a fluidic conduit 4305 isconnected to the partitioner 4304 by a compression fitting using a nut4314 and ferrule 4315. The fitting generates a fluidic seal between thepartitioner and tubing as the fitting is tightened between the outersurface of the tubing and the inner surface of the partitioner. Theinternal cross-sectional diameter of the tubing 4317 may match theinternal cross-sectional diameter of the conduits in the partitioner4318 to ensure smooth fluid flow through the connection. The fittingsmaterial may be made out of any suitable material as long as thatmaterial is appropriate for the continuous phase solvents being used. Ina second embodiment, channels are generated in a substrate 4304 with twodistinct inner diameters. The first inner diameter 4318 is of ample sizeto allow flow of fluid to the partitioner junction 4319 and may be ofequal size to the fluidic conduit being connected 4317 to ensure smoothflow through the connection. The second inner diameter 4320 is of a sizeto connect the fluid conduit through an interference fit. The diameterof second inner diameter 4320 is equal to or smaller than the outerdiameter of the fluidic conduit to be inserted. When inserting a fluidicconduit 4305 made with a material of a higher elastic modulus than theblock material, a sufficient force is applied to the tubing forming afluidic seal.

The droplet partitioner may or may not be mounted using a supportingmaterial 4316. The mounting material may be composed of any materialcapable of machining, engraving, etching, ablating, embossing, molding,or printing. The mounting material may be used to affix and stabilizethe partition in a specified location in the system. The material mayalso be made of a conductive material to help dissipate any static buildup at the partitioner. It may also be used to stabilize conduitconnections to the partitioner. For example, when using an interferencefit connection, the fluidic seal is generated inside of the partitionerat the interface of the inner surface of partitioner and the outersurface of the fluidic conduit. However, additional support may berequired to further secure the union from disturbance and may begenerated using a nut and ferrule assembly 4314 connected to asupporting mount 4316.

The channels in the partitioner may be generated through the use oftubing and/or drilled, milled, or otherwise formed channels in a solidblock. Suitable characteristic dimension, e.g., diameter for circularchannels, range from 50 um to 2 mm, such as 50 um to 1 mm, or 50 um to500 um, or 100 um to 400 um, or 200 um to 2 mm, or any other range ordimension as described herein in specific embodiments. When tubing isused, the tubing is placed sufficiently into the block, e.g.,fluoropolymer block to act as fluidic channels at the junction. Whenformed channels are used, the tubing is placed adjacent to drilledchannels in the fluoropolymer block. Any suitable combination of fluidicchannel types may be used, for inlet channels and/or outlet channel.

In certain embodiments, fluoropolymer tubing is inserted into thefluoropolymer block adjacent to formed channels, e.g., drilled or milledchannels equal to or smaller than the tubing ID. The inlet and outletchannels can be produced in the fluoropolymer block in two passes. Inthe first pass, a channel is generated to accommodate either theinterference or the compression forming a fluidic seal with the tubing.In the second pass, the liquid channel is generated.

The droplet partitioner channels in the fluoropolymer block are madesufficiently small to reduce the possibility for hold up of sample inthe channels.

Channel diameters for the tubing (or other appropriate conduit)connecting to the inlet channels can take any dimension. However, it isgenerally preferable to avoid transitioning from smaller diameters to alarger diameter in the droplet partitioner inlet channels, because itcan potentially cause breakup of the inlet train of dispersed phase(e.g. aqueous PCR reactions), holdup, sample adsorption or otherproblems that lead to, e.g., cross-contamination between samples and/orinaccurate readings for a sample due to partial removal of part of thesample. Also, there is little benefit to being very small in theconnection conduits, for pressure drop reasons. Generally, thecharacteristic dimension, e.g., diameter, of these inlet conduits canbe, at least 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, or 450 um,and/or not more than 75, 100, 125, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, or 800 um, for example, between 75 and 800 um, or between125 and 700 um, such as between 150 um and 500 um.

Connection to the outlet channel generally follows the same pattern. Incertain embodiments, a conduit leading from the partitioner to the restof the system can have a characteristic (critical) dimension, e.g., adiameter, of at least 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, or450 um, and/or not more than 75, 100, 125, 150, 200, 250, 300, 350, 400,450, 500, 600, 700, or 800 um, for example, between 75 and 800 um, orbetween 125 and 700 um, such as between 150 um and 500 um.

Thus, systems and methods as described herein may comprise reducingcontamination by of dead zones. “Dead zones” as described herein referto regions or zones in the flow where droplets of the dispersed phase orcontinuous phase have low or zero velocity. In some instances, thesystem comprises multiple microfluidic tubes, channels, or modules thatare interconnected to provide a flow pathway for the continuous phase,emulsion and/or dispersed phase. These interconnections may be locationsat which dead zones could form. For example, an interconnection may bewhere there is an imperfect connection between one microfluidic tube ormodule and a second microfluidic tube or module, and a dead zone canform in the imperfect connection. Dead zones can lead tocross-contamination because a first portion of a first dispersed phasegenerated from a first sample may be trapped in the dead zone longenough to contact or be interspersed with a portion of a first dispersedphase generated from a second sample. To prevent formation of deadzones, one or more connections may be made between the one or moremicrofluidic channels or one or more tubes in the system. For example,one or more connections are made between the microfluidic channels orthe tubes of the injector, droplet generator, reactor, or detector. Forexample, a press-fit connection can be made between a microfluidic tubeand a microfluidic channel (a first conduit and a second conduit). Thepress-fit connection may comprise a recessed region in a polymer leadingto the microfluidic channel. The cross-section of the microfluidicchannel (first conduit) may substantially match the internalcross-section of the tube (second conduit). In some instances, themicrofluidic channel and the microfluidic tube comprise differentcross-sections. In some instances, an internal cross-section of therecessed region is smaller than the external cross-section of the tube.In some instances, an internal cross-section of the recessed region isthe same as the external cross-section of the tube. The tube maycomprise a material with a lower elastic modulus than the polymercontaining the channel. Exemplary materials of the tube include, but arenot limited to, silicon, polystyrene, polyacrylamides, PDMS, ceramic,metals, and glass. Exemplary polymers of the channel include, but arenot limited to, a styrenic elastomer, an ethylene vinyl acetateelastomer, a polyolefin elastomer, a diene elastomer, fluoropolymers(e.g. PTFE, PVDF, PFA, and FEP), or combinations thereof.

Provided herein are systems and methods for serial flow emulsionreactions, wherein reducing contamination by preventing formation ofdead zones may comprise creating a seal. In some instances, as a resultof a connection between the tube and the polymer, a fluidic seal iscreated. In some instances, the fluidic seal prevents continuous flow ofdroplets.

In some instances, placement of the tube prevents creation of a deadzone. For example, placement of an end of the tube against an end of therecess prevents creation of a dead zone and a continuous flow. Acontinuous flow may also be a result of the cross-section of the channeland the cross-section of the tube being substantially the same.

Fabrication of partitioners. For partitioners described herein, such asreverse-y, T-partitioners, or flow-focusing partitioners, any suitablefabrication method may be used; for convenience, methods will bedescribed for the reverse-y partitioner but it will be appreciated thatT-partitioners and other design can employ the same or similartechniques.

Droplet partitioners can be fabricated from any suitable materials ofconstruction, including glass, polymers, metal, ceramics, or any mixtureof these. Any suitable fabrication method that creates the desiredfeatures, e.g., removes material from a solid piece or builds upmaterial to have the specified feature geometries, may be used.Fabrication techniques include but are not limited to direct machining,engraving, etching, ablating, embossing, molding, printing.

Provided herein are methods of fabricating partitioners, e.g., from asolid block of material, or from a plurality of blocks joined togetherto provide a fluidically tight seal. In certain embodiments, the blockor plurality of blocks comprise material that has greater affinity forcontinuous phase to be used in the system than for dispersed phase—thisprovides an easy and rapid method to manufacture a partitioner with thedesired properties without requiring coating or otherwise altering thesurface and produces partitioners that will continue to functionproperly even if the surfaces are subject to wear.

In some cases, suitable channels may be created by creating holes, e.g.,drilling into a solid block. For example, for a reverse-y partitioner ina configuration such as shown in FIG. 44, where the inlet channels meetat 180 degrees, or any other type of partitioner where conduits of thesame diameter meet at 180 degrees, a single drilling or similaroperation can produce both inlet channels and a second drillingoperation can produce the outlet channel; in the case of a partitioneras shown in FIG. 39, more than 2 drilling operations can be required. Ifpress fittings or compression fittings are used, a larger hole toaccommodate the tubing may be drilled first, then the smaller-diameterhole that will comprise the channel can be drilled. Suitable machiningor other operations may be used to produce a junction of the larger withthe smaller conduit that will butt firmly with tubing inserted into thelarger-diameter conduit. Thus, in certain embodiments, the first andsecond inlet channels are oriented 180 degrees apart, so that a singledrilling, milling, or other similar pathway is used to produce thesechannels. The outlet channel is created by a drilling, milling, or othersimilar operation that connects with the inlet channels. Each inlet oroutlet channel may be a single drilled fluidic channel, a single drilledtubing channel, or a combination of the two.

However, other manufacturing procedures may be used. See, e.g., FIGS.46A and 46B. FIG. 46A shows a combination of milling and drilling. Thedrill step creates the outlet. The mill step creates the two inlets.They may be at any angle from each other from 2-180 degrees (90 degreesis shown in the Figure). Once the two pieces are made they are joined byany suitable method, for example, fused together chemically or are heldtogether tightly to create a fluidic seal. The first method ispreferred. FIG. 46B shows a 3 piece assembly generated entirely bymilling. The 3 pieces are then fused together. Any suitable number ofpieces may be drilled or milled, then joined, such as at least 2, 3, 4,or 5 pieces. The surface chemistry of the channels after bonding shouldbe consistent with the chemistry of the continuous phase. With ahydrophobic continuous phase the material should be hydrophobic; for anaqueous continuous phase and the material being hydrophilic. Forexample, if fabrication begins with a fluoropolymer material, the blocksmay be sealed together, and the channels are still composed offluoropolymer, thus simplifying manufacturing. If a chemistry is usedthat destroys the fluoropolymer surface, it can be restored to befluorinated. In embodiments in which the partitioner is a block that iscomposed of material with higher affinity for the continuous phase thanfor the dispersed phase, e.g., a fluoropolymer block, there is a benefitthat if there surface is degraded/etched over time, instead of losingthe fluoropolymer surface, new fluoropolymer is exposed. This ensuresthat the surface chemistry of the partitioner is more robust thantraditional PDMS/glass droplet partitioners coated with a fluoropolymer.

Thus, while the material of construction is not necessarily importantfor droplet formation, it may be desirable for the surface of thepartitioner to comprise a material with a higher affinity for thecontinuous phase than for the dispersed phase; droplet partitionersconstructed of materials with substantially similar affinities for thetwo phases or a higher affinity for the dispersed phase than for thecontinuous phase may be coated with a film comprising a material with ahigher affinity for the continuous phase than for the dispersed phase onthe appropriate surfaces, e.g., at least on surfaces that will contactthe dispersed phase. This film may, over time, chemically or physicallydegrade, exposing the underlying surface to the emulsion and disruptingdroplet partitioning or holding up a portion of the dispersed phase.This can affect droplet size distribution, stability, andcross-contamination in negative ways. For example, a droplet partitionercould be constructed of glass with a surface fluoropolymer coating, thecontinuous phase comprising a fluorinated oil, and the dispersed phasecomprising water. This partitioner's coating may wear over time. Ifinstead, the entire droplet partitioner was made of fluoropolymer,chemical or physical degradation of the surface would only reveal morefluoropolymer maintaining the intended surface properties. Thus, allother elements being equal, it is desirable that the droplet partitionerhave a thick layer comprising a material with higher affinity for thecontinuous phase than for the dispersed phase. In a preferredembodiment, the droplet partitioner channels themselves comprise such amaterial.

Surfaces of the droplet partitioner that come in contact with sample canbe composed of materials compatible with biological assays. Suchmaterials do not interfere with biochemical reactions, do not havesignificant affinity for biological materials or biochemical reagents,and/or do not substantially destabilize the emulsion of droplets orinterfere with droplet formation. General classes of these materialsinclude thermoplastics, silicones, fluoropolymers. In certainembodiments, these materials comprise a fluoropolymer.

The manufacturing methods available to producing microfluidic devicesout of fluoropolymer are limited. One method is to machine thefluoropolymer to create the first inlet channel and the second inletchannel as well as the outlet channel. In certain embodiments, the firstinlet channel is created by a drilling, milling, or similar operation,the second inlet channel is created by a drilling, milling, or similaroperation, and the outlet channel is created by a drilling, milling orsimilar operation. In certain embodiments, the first and second inletchannels are oriented 180 degrees apart, so that a single drilling,milling, or similar pathway can be machined to produce these channels.In this embodiment, the drilled channels are the liquid channels. Whenmachining at this scale (<300 um), the length of channels that can becreated can be limited by the length of tooling available. In certainembodiments, one or more of the drilled channels are produced to supporta fluidically tight seal to an inserted tubing. In this instance, theinserted tubing forms the liquid channels. The characteristic dimension,e.g., diameter, of the inserted outlet tubing determines the dropletdiameter.

In certain embodiments, such as that shown in FIG. 46A the first inletchannel 4602 and the second inlet channel 4603 can created by at leastone milling or similar operation on a substrate 4605, e.g.,fluoropolymer surface, at least one debossing or similar operation on asubstrate, e.g., fluoropolymer surface, or a combination thereof. Theoutlet channel 4601 can be created by at least one milling, drilling orsimilar operation into a material 4604, e.g., comprising a fluoropolymerso that the surface of the channel is fluoropolymer. The dropletpartitioner is created by fusing the material containing the outletchannel to the fluoropolymer surface containing the first and secondinlet channels so that a fluidic seal is created outside of the first4602 and second 4603 inlet channels and the outlet channel 4601. Methodsof fusing are as described herein. In another embodiment (FIG. 46B), thepartition is generated from three or more pieces. The first inletchannel 4602 and the second inlet channel 4603 are created by at leastone milling operation in a first substrate 4607, e.g., comprising afluoropolymer surface, at least one embossing operation on a, e.g.,fluoropolymer surface, or a combination thereof. The outlet channel iscreated by at least one or more milling operations into a first material4607 comprising a fluoropolymer forming a portion of the outlet channel4608 and a second material 4609 forming another portion of the outletchannel 4610. The droplet partitioner is created by fusing the materialcontaining the outlet channel to the fluoropolymer surface containingthe first and second inlet channels so that a fluidic seal is createdoutside of the first 4602 and second 4603 inlet channels and the outletchannel 4603. This fluidic seal creates a junction 4606 where twofluidics meet and exit via the outlet channel 4601. Methods for fusingare as described herein.

In embodiments where two or more pieces are fused, any suitable methodfor fusing the materials may be used. Exemplary methods for fusing thesematerials together include fusion welding, ultrasonic welding, heatwelded or chemical bonding. Chemical bonding requires etching thefluoropolymer surface to make it amenable to chemical bonding agents.The channel features may be generated before or after etching. In thefirst instance where the channels are generated before etching, both thesurface to be bonded and the channels are no longer fluorinated. Afterbonding, the channels may once again be rendered fluorinated by chemicaldeposition. In the second instance where the channels are milled orotherwise created after etching, the bonding restores the channelmaterial to its native properties. In some cases, the pieces may bephysically crimped or clamped together if sufficient force is applied togenerate a fluidically tight seal.

Connections of the partitioner with conduits to the rest of the systemmay be made as described herein, e.g., by press fit or compression fit,or other suitable method to provide a connection that does not disruptfluid flow to a significant degree, e.g., to a degree where a deadspace, eddy, swirl, or other disruption is formed.

In certain embodiments, fluoropolymer tubing is inserted into thefluoropolymer block adjacent to drilled or milled channels equal to orsmaller than the tubing ID. The inlet and outlet channels are producedin the fluoropolymer block in two passes. In the first pass, a channelis generated to accommodate either the interference or the compressionforming a fluidic seal with the tubing. In the second pass, the liquidchannel is generated.

The droplet partitioner channels in the fluoropolymer block are madesufficiently small to ensure stable fluid flow reducing the possibilityfor hold up of sample in the channels.

In certain embodiments, the tubing ID and channel ID are size matched.

Concentrating droplets (disengagement). In certain embodiments, afterpartitions are formed at the partitioner, a portion of the continuousphase (e.g., oil, such as a fluorinated oil) is removed from theemulsion (disengagement), thus concentrating the droplets. The systemfor removing continuous phase and, in certain embodiments, addingremoved continuous phase back to the system is referred to herein as adisengager. This can be useful to slow the fluidic velocity, since thereis not as much liquid moving through the system at any given time. Lowfluidic velocity can improve droplet stability and reduce the effect ofunstable liquid flows that can induce droplet breakage,cross-contamination, droplet transit between samples, and the like. Incertain embodiments, an amount of continuous phase is removed sufficientto slow fluidic velocity by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 12, 15, 17, 20, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, or 85% of the fluidic velocity before continuous phase removal,and/or at most 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 22,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% of thefluidic velocity before continuous phase removal. In certainembodiments, the amount of continuous phase removed is at least 0.1,0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 22, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, or 85% of the continuous phase, and/orat most 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 22, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% of the continuousphase, such as continuous phase as described herein, e.g. an oil, forexample a fluorinated oil, for example, 20%-80%, such as 30-70%, or30-99%, or 80-99.5%, or 90-99.5%. It is desirable to remove continuousphase in such a way that droplets are not also removed, e.g., in such away that less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.8, 0.6, 0.5, 0.4,0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001% of dropletsare also removed. In certain cases, an impediment to droplet movementwith the continuous phase, such as a filter, is used to ensure thatdroplets are not removed with the continuous phase. In certain cases,some or all of the removed continuous phase can be re-used, for example,to separate droplets prior to detection (see below); at the point orpoints of reintroduction, there may also be a second impediment, such asa filter, to ensure that droplets do not move into the continuous phaseconduit. Some or all of the removed continuous phase may be returned tothe system; e.g., continuous phase removed after the partitioner andbefore the reactor, e.g., thermal cycler, may be returned to the systembefore the interrogation region of the detector in order to separatepartitions; for example, 10-100% of removed continuous phase may bereturned, such as 50-100%, for example, 80-100%.

Removal of continuous phase can be active or passive. If the removal isactive, any suitable method for producing a force to move droplets orcontinuous phase may be used. FIG. 47A illustrates active removal usingan electrical feature that pushes or pulls droplets from one channel toanother (dielectrophoretic sorting). Passive movement, as shown in FIG.47B, can use fluidic resistance and/or buoyancy to prefer upward dropletmovement in an embodiment of a system for reducing the amount ofcontinuous phase fluidic between dispersed phase partitions. In FIG.47A, the system comprises a substrate that comprises a main channel4701, a first outlet channel 4702, and a second outlet channel 4703. Thetwo outlet channels connect to the main channel at a commonintersection. In this embodiment there is also an electrode thatgenerates a non-uniform electric field 4704 and any grounding electrodesrequired. Loosely spaced partitions enter the main channel 4701, enterthe non-uniform electric field 4704 and are moved towards the firstoutlet channel 4702 through dielectrophoretic force. The relative amountof continuous phase fluid that flows to the first 4702 and second 4703outlet channels is based on the relative flow rates between the twochannels. Droplets exiting through the first outlet channel 4702 aremore packed than the droplet entering the main channel 4701. FIG. 47Bshows another embodiment of a system for reducing the amount ofcontinuous phase fluidic between dispersed phase partitions. The systemcomprises a substrate that comprises a main channel 4701, a first outletchannel 4702, and a second outlet channel 4703. The two outlet channelsconnect to the main channel at a common intersection. In this embodimentthe planar orientation of the channels is parallel to the gravitationalforce 4705. In orienting the substrate in this manner, gravitationalforce 4705 and buoyance may play a part in droplet mobility. Looselyspaced partitions enter the main channel 4701, float towards the top ofthe conduit, and transit towards the first outlet channel 4702. Sincethe droplets are positioned at the top of the channel and the firstoutlet channel 4702 is present at the top of the substrate, the dropletspreferentially exit through the first outlet channel 4702. The relativeamount of continuous phase fluid that flows to the first 4702 and second4703 outlet channels is based on the relative flow rates between the twochannels. Droplets exiting through the first outlet channel 4702 aremore packed than the droplet entering the main channel 4701.

Continuous phase can be removed at any suitable location after thepartitioner, and can be removed at 1, 2, 3, or more than 3 locations.Continuous phase that has been removed can also be reintroduced at anysuitable point, before or after the point of removal, or both, and canbe reintroduced at 1, 2, 3, or more than 3 locations. In certainembodiments, continuous phase is removed after droplets have passedthrough a detector. See FIG. 48. Some or all of the removed continuousphase can then be reintroduced into the system at one or more suitablelocations; FIG. 48 shows continuous phase (oil) reintroduced at a pointbefore the detector, in order to separate partitions as they enter thedetection zone (see description of detection, below). The systemcomprises a T-connector 4802 connecting a main conduit 4801 flowingdensely packed partitions, a second conduit flowing additionalseparation fluid, such as oil 4803, a physical barrier with pore sizessignificantly smaller than the partition diameter 4804, and an outletconduit 4805. The outlet conduit comprises a constricted region 4814. Afirst continuous phase and partitions of dispersed phase enter the mainconduit 4801 while a second continuous phase miscible with the firstcontinuous phase enters through the second conduit 4803. The additionalcontinuous phase fluid is injected into the conduit 4803 from areservoir 4812 using a pump 4813. Upon exiting the T-connector 4802, theaverage separation of the dispersed phase partitions (average distancebetween adjacent partitions) is greater than upon entering theT-connector. The spaced partitions enter the constricted region of theconduit 4814 and enter the interrogation region 4806 where a parameterof the partitions is interrogated. Spaced partitions enter a secondT-connector 4810 through a conduit 4807. The partitions enter a branchedconnection. Continuous phase fluid flows through both the first outletconduit 4811 and the second outlet conduit 4808 based on the relativeflow rates through each conduit. Partitions are unable to enter thefirst outlet conduit 4811 due to the physical barrier generated by 4809.Since partitions are unable to enter the first outlet conduit 4811 andflow preferentially towards the second outlet conduit 4808, thepartitions are more densely packed then when they were in theconstricted region 4814. The additional continuous phase fluid enteringthe first T-connector 4802 is recycled upon reaching the secondT-connector 4810.

In certain embodiments, continuous phase is removed after droplets havebeen formed at the partitioner but before they move through a reactor.See FIG. 49, in which continuous phase is removed prior to a cycler(e.g., thermal cycler, such as for PCR). Some or all of the removedcontinuous phase can then be reintroduced into the system; FIG. 49 showscontinuous phase (oil) reintroduced at a point before the detector, inorder to separate droplets as they enter the detection zone (seedescription of detection, below). The system comprises a T-connector4902, a main conduit 4901 flowing sparsely packed partitions, a firstoutlet conduit 4903 in which continuous phase fluid may flow, a physicalbarrier with pore sizes smaller than the partition diameter 4904, and asecond outlet conduit 4905 flowing densely packed dispersed phasepartitions. The outlet conduit leads to the larger partition handlingsystem 4914. At the junction, continuous phase fluid may flow throughboth outlets based on the relative flow rates, but partitions may onlyflow towards the second outlet conduit 4905 due to the physical barrier4904. As a result, the originally loosely packed partitions are moredensely packed after transiting through the first T-connector system.Upon transiting the droplet handling system 4914, densely packeddispersed phase partitions enter a second T-connector 4910. The secondT-connector comprises a first inlet conduit 4911, a physical barrierwith pore size smaller than the partition diameter 4909, and a secondoutlet conduit 4908 that leads to a conduit constriction 4912. Theadditional continuous phase fluid exiting the first T-connector entersthe second T-connector through the first inlet conduit 4911. The averagespacing of the densely packed droplets entering the main inlet conduit4907 of the second T-connector 4910 is increased as the transit thefirst inlet channel 4911. The loosely packed droplets enter the firstoutlet conduit 4908, enter a constricted region 4911. The spaceddroplets enter the interrogation region 4906 where one or moreparameters of the partitions are interrogated.

It will be appreciated that the embodiments exemplified in FIG. 48, andFIG. 49 can be combined in any suitable manner.

Similar construction and connection methods may be used for thedisengagers as described for partitioners, with suitable modificationsto account for, e.g., filter elements and the like.

In some instances, a system comprises a rotating pin. The rotating pinmay have a cavity that is partially open. When the system is“collecting” droplets from a droplet generator, the droplets may move upor down into the cavity where the droplets are trapped at the roof orfloor of the cavity. Once the droplets are all collected, the pin mayrotate and the cavity is exposed to an open channel in a second part ofthe device, where another continuous flow input drives the dropletsthrough the thermal cycler and detector. Once all of the droplets havepassed the channel, the rotating pin may rotate back around and is readyto accept a new dispersed phase comprising droplets.

B. Reactor

The systems and methods provided herein can include moving partitionsformed by the partitioner through a reactor. The reactor can be anysuitable reactor that initiates and/or modulates one or more desiredreactions in one or more of the partitions. Thus, a reactor can be areactor that introduces energy, e.g., thermal energy, electromagneticenergy, acoustic energy, or other suitable energy, to partitions as theyflow from the partitioner.

In certain embodiments, the reactor comprises a thermal cycler, e.g., athermal cycler for cycling partitions through a plurality of temperaturezones to cycle PCR reactions. Any suitable configuration may be used solong as it serves to expose partitions to temperature zones that areconducive to different parts of the PCR cycle. Generally, a conduit,such as a tube, is used to allow flow of partitions. In certainembodiments, the conduit is in contact with, e.g., wrapped around, acylindrical or substantially cylindrical core that comprises at least 2zones of different temperatures. The temperature zones may allow for ahot start reaction, thermal cycling, annealing, extension,polymerization, or protein denaturation. The conduit, e.g., tubing, thusforms a helical structure or substantially helical structure.

It can be desirable to minimize buoyancy effects along sections of theconduit, e.g., at least in sections of the conduit in the thermalcycler. In certain embodiments, the radius of curvature and the rate offlow of the partitions in the conduit are in a range where flow in theconduit is laminar or substantially laminar. In certain embodiments,most or all of the conduit, for example, most or all of the conduit fromjust after the partitioner to the detector, can be kept in a plane thatis orthogonal or nearly orthogonal to gravity; it will be appreciatedthat certain sections, such as coils around a racetrack thermal cycler,must, of necessity, deviate somewhat from the orthogonal plane but ingeneral will remain nearly orthogonal to the plane. Thus, in certainembodiments, the conduit, such as the conduit from just after thepartitioner to the detector, is configured so that no section of theconduit, or no significant section of the conduit, deviates from a planethat is orthogonal to gravity by more than 30, 20, 15, 10, 7, 5, 4, 3,2, or 1 degree, where the angle is measured from the axis of flow in theconduit to the plane orthogonal to gravity. It will be appreciated thatsmall sections of the conduit may deviate more than this from theorthogonal plane, e.g., up to 1, 2, 3, 4, 5% of the conduit, so long asthe deviation does not produce significant buoyancy or other effectsthat lead to undesirable consequences, e.g., axial spreading ofpartitions to such a degree that one group of partitions may merge withanother

FIG. 76 shows a heater-reactor conduit. An embodiment of a system forproviding distinct zones where droplets can be maintained at particulartemperatures for set times FIG. 76. The system comprises a conduit thattraverses one or more core heater elements that are maintained at aconstant temperature. A core heater element is a block of material thatis held at a specific temperature. The construction of a core heaterelement is such that it has a glass transition or melting temperature ofat least 120, 200, 500 degrees Celsius. In a preferred embodiment thecore heater elements are constructed out of a solid block of metal, forexample aluminum, that has a high thermal conductivity, for example athermal conductivity of more than 50, 100 or 200 W/m K. The temperatureof the core heater elements is controlled with resistive heatingelements. When current is applied across the resistive heating elements,they conduct heat into the core heater elements. Resistive temperaturesensors are used to measure the temperature of the core heater elements.In some embodiments the resistive temperature sensors are thermallypotted into the core heater elements to provide the most accuratemeasurements of the core heater element temperature. A control system isapplied to read the temperature from the resistive temperature sensorsand control the amount of current applied to the resistive heatingelements. The control system is designed to hold the temperature of thezone within 2, 1, 0.5, 0.1 or less than 0.1 degree Celsius. The coreheater elements are individually isolated to minimize conduction betweenthe elements. The heated core elements of the heater-reactor can be flator curved and the number of elements can vary from a single element toat least 3, 4, 5, 10, 20, 100 elements. The size and number of the coreheater elements is used to determine the time a particular droplet thatpasses through the conduit of the heater reactor is held at specifictemperatures. In a particular embodiment a single core heater element isa hollow cylinder that has a fluid conduit at the outer surface. In thisexample the diameter of the cylinder, the number of wraps of theconduit, and the fluid velocity in the conduit help determine the timeeach droplet spends at the desired reaction temperature. In certainembodiments where a single core heater element is used the system isdesigned to process dispersed phase samples for isothermal reactions. Inother embodiments multiple core heater elements are combined such thatthey form a cylindrical shape that a conduit can wrap around. Inembodiments where more than one core heater element is used the systemcan be designed to process dispersed phase samples for one or moreisothermal reactions or for reactions that require temperature cycling.Some isothermal reactions may require two or more core heater elements.

In certain embodiments the conduit comprises tubing 7601 that enters atthe bottom of the assembly where the conduit is held in place with aretaining system 7602 clamps the tubing to fix it in position but doesnot change the outer or inner diameter of the tubing by more than 1%,2%, 5%, or 10%. The conduit is then wrapped around the exterior of thecore heater element or elements 7603. The conduit leaves the top of theassembly where it is held in place with a retaining system 7604 clampsthe tubing to fix it in position but does not change the outer or innerdiameter of the tubing by more than 1%, 2%, 5%, 10%. The conduit thencontinues 7605 until connects to the next subunit in the instrument,such as a detector assembly. In some embodiments the core heaterelements of the heater-reactor are held in place by structural elementsthat have low thermal conductivity 7606 and 7607, for example a thermalconductivity of less than 5, 1, 0.1 or 0.01 W/m K.

In some embodiments the cross-sectional area of the tubing is such thatdroplets must travel in a single file and there is insufficient room fordroplets to pass each other. The diameter of the conduit may be larger,smaller, or equal to the equivalent spherical cross-sectional diameterof the partitions flowing through the channel. In some embodiments thecross-sectional area allows two droplets to move through the tubing sideby side. In some embodiments the cross-sectional area allows more thantwo droplets to travel through the tubing side by side. In a preferredembodiment, the diameter of the conduit is larger than the equivalentspherical diameter of the dispersed phase partitions flowing through theconduit but is not large enough to have more than three full equivalentspherical diameters of the dispersed phase partitions flowing through across-section of conduit at a time. If the conduit through theheater-reactor is in direct fluid communication with the partitioner,then the fluid flow rate through the conduit is equal to flow rateleaving the partitioner and the rate remains constant throughout theconduit in the heater-reactor. In some embodiments a continuous phaseflows through the heater during normal system function. When a firstdispersed phase is partitioned in the partitioner it then moves throughthe conduit of the heater-reactor. In some embodiments the conduit isconstructed out of flexible tubing that comprises a thermoplasticmaterial such as polyolefins, polyurethanes, fluoropolymers, or blendsof like materials. In a further embodiment the conduit comprises amaterial that has a higher affinity for the continuous phase than forthe dispersed phase, such as PTFE, PFA, FEP or other like materials.

Radiant thermal energy from the heater-reactor is released to thegreater system through convection. Air flow through the instrument isable to remove heat from the instrument the to help maintain a stableinstrument operating temperature. The stable instrument operatingtemperature is below the temperature of the heater-reactor so that theheater-reactor can cool by convection. In some embodiments insulation isapplied to exposed surfaces of the heater-reactor to control the rate ofconvective heat transfer. The insulation comprises materials that havelow thermal conductivity for example a thermal conductivity of less than5, 1, 0.1 or 0.01 W/m K.

In some embodiments more than one conduit can be wrapped around the sameheated core materials. Such an embodiment provides the opportunity for afirst dispersed phase, dispersed phase 1 to travel down one conduit,conduit A while the subsequent dispersed phase, dispersed phase 2 isdirected down the second conduit, conduit B. Each subsequent dispersedphase that is added to the process side. The ability to switch theconduit can decrease the time between dispersed phase injections.

In some embodiments more than two conduits are wrapped around the sameheated core materials. Such an embodiment provides the opportunity toswitch the conduit path for each dispersed phase that is added to theprocess side.

In some embodiments where two or more conduits are wrapped around thesame heated core materials, the individual conduits follow a helicalpath. In some embodiments the cross-sectional geometry of the conduit iscircular, square, rectangular, triangular, oval, or some combination ofthese shapes.

In some embodiments the radius of curvature of the conduit is maximizedto prevent the shear forces exerted on the partitions from exceeding theinterfacial tension stabilizing the partition surface. In someembodiments the vertical rise of the conduit is minimized to reduce theability of buoyant forces to move partitions relative to each otherwithin a single dispersed phase sample. In some embodiments the fluidvelocity helps to reduce motion of partitions relative to each otherwithin a single dispersed phase sample.

FIGS. 77A and 77B show a heater-reactor conduit An embodiment of aheater-reactor system for providing one or more distinct zones wheredroplets can be maintained at particular temperatures for one or moreset times FIG. 77. The system comprises a conduit that travels from thedispersed phase partitioner through the heater-reactor and on to thedetector assembly. In a particular embodiment a single core heaterelement is a hollow cylinder with the fluid conduit wrapped around outersurface. In some embodiments the number of wraps of the conduit can varyto change the amount of time the partitions spend at a particulartemperature. The diameter of the cylinder can also be adjusted to varythe residence time of the partitions within the heater-reactor. In apreferred embodiment the core heater element has grooves (FIG. 77B) thatcapture the conduit and increase the contact surface area between theconduit and the core heater element. In some embodiments the groovedepth allows at least 5, 10, 25, 50, 75 or 100% of the conduit to becaptured. In some embodiments the groove is deeper than the diameter ofthe conduit such that the conduit is entirely captured and sits belowthe outer surface of the core heater element.

FIG. 78 shows a three temperature zone heater-reactor FIG. 78 shows anembodiment of a heater-reactor that contains three distinct core heaterelements. Each of the core heater elements is independently controlledso that it is maintained at a particular temperature. In someembodiments the temperature of each core heater element is distinct. Inother embodiments two or more of the core heater elements are maintainedat the same temperature. In some embodiments the core heater elementsare assembled so that they form a cylindrical shape. In some embodimentsthe conduit is wrapped around the core heater elements such that itforms at most a single 360 degree wrap around each core heater element.In some embodiments the conduit is wrapped around the core heaterelements such that it makes more or more passes across each of the coreheater elements.

FIG. 79 shows a four temperature zone heater-reactor. FIG. 79 shows anembodiment of a heater-reactor that contains four distinct core heaterelements. Each of the core heater elements is independently controlledso that it is maintained at a particular temperature. In someembodiments the temperature of each core heater element is distinct. Inother elements two or more of the core heater elements are maintained atthe same temperature. In some embodiments the core heater elements areassembled so that they form a cylindrical shape. In some embodiments thecore heater elements are flat and the conduit passes back and forthacross the elements in a serpentine arrangement. In some embodimentswhen the core heater elements are flat they at positioned perpendicularto the gravitational field so that the buoyant force is perpendicular tothe fluid velocity to minimize the impact of buoyancy impacting thevelocity of a partition within the conduit.

FIG. 80 shows a discrete four temperature zone heater reactor showsanother embodiment of a heater-reactor that contains four distinct coreheater elements that are arranged in distinct sub-assemblies. Each ofthe core heater elements is independently controlled so that it ismaintained at a particular temperature. In some embodiments thetemperature of each core heater element is distinct. In otherembodiments two or more of the core heater elements are maintained atthe same temperature. In some embodiments the core heater elements areassembled so that they form a cylindrical shape. In some embodiments thecore heater elements are flat and the conduit passes back and forthacross the elements in a serpentine arrangement. In some embodiments thecore heater elements are assembled such that each distinct sub-assemblyof core heater elements can be either flat or cylindrical. In someembodiments a valve is included such that partitions can either passthrough all of the core heater element sub-assemblies or only through asub-set of these sub-assemblies.

FIGS. 81A, 81B, and 81C show a gradient heater-reactor FIGS. 81A, 81B,and 81C show exemplary layouts for core heater elements that can beincluded as part of a heater-reactor. Each of the core heater elementsis independently controlled to maintain a temperature profile that canbe a distinct temperature or a gradient of temperatures. In someembodiments one or more core heater element can have a horizontaltemperature gradient where there is a portion of the core heater elementthat is maintained to the highest desired temperature and a gradient oftemperatures is formed where the rest of the core heater element has alower temperature. In some embodiments this gradient is controlled byapplying heat to particular portion of the core. The core is designedwith materials that have a thermal conductivity to enable the formationof controlled gradient. In some embodiments one or more core heaterelement can have a vertical temperature gradient. As partitions ofdispersed phase samples move through a conduit that is in thermalcontact with the core heater element or elements, they are maintained ata temperature that matches the gradient of the core element. Such anembodiment enables the temperature of the partitions to be transitionedat controlled rates that might be critical for particular reactions tobe performed efficiently. In some embodiments a large number of discreetcore heater elements are arranged in a horizontal or vertical, or both,orientation to create a large number of distinct temperature holds forthe partitions.

FIGS. 82A and 82B show a 2-step PCR heater reactor. FIG. 82 showsexemplary layout for a heater-reactor with three core heater elements.The system comprises a conduit that travels from the dispersed phasepartitioner through the heater-reactor and on to the detector assembly.In a particular embodiment a single core heater element is a hollowcylinder with the fluid conduit wrapped around outer surface. In aparticular embodiment the lowest core heater element is maintained at anelevated temperature of 95 degrees Celsius to act as an initialdenaturation cycle for a PCR reaction. In this example zone two is alsoheld at 95 degrees Celsius to provide further denaturation cycles andzone three is held at a temperature less than 95 degrees Celsius toprovide an annealing and extension cycle for a PCR reaction. In thisexample the conduit is wrapped around zone one at least once and iswrapped around zones two and three at least 24, 28, 32, 36 or 40 timesto provide that number of thermal cycles for the partitions totransition between a denaturing temperature and an annealing/extendingtemperature. The size of zones two and three are designed to control thetiming of the denaturing and annealing/extension cycles.

FIGS. 83A and 83B show a RT-PCR heater-reactor. FIG. 83 shows exemplarylayout for a heater-reactor with four core heater elements. The systemcomprises a conduit that travels from the dispersed phase partitionerthrough the heater-reactor and on to the detector assembly. In aparticular embodiment a single core heater element is a hollow cylinderwith the fluid conduit wrapped around outer surface. In a particularembodiment the lowest core heater element is maintained at a temperatureoptimized for reverse transcription reactions. As the conduit movesvertically the next core heater element it comes in contact with is heldat an elevated temperature of 95 degrees Celsius to act as an initialdenaturation cycle for a PCR reaction. In this example zone two is alsoheld at 95 degrees Celsius to provide further denaturation cycles andzone three is held at a temperature less than 95 degrees Celsius toprovide an annealing and extension cycle for a PCR reaction. In thisexample the conduit is wrapped around zone one at least once and iswrapped around zones two and three at least 24, 28, 32, 36 or 40 timesto provide that number of thermal cycles for the partitions totransition between a denaturing temperature and an annealing/extendingtemperature. The size of zones two and three are designed to control thetiming of the denaturing and annealing/extension cycles.

Thus, following generation of droplets, the droplets may flow to areactor. In some instances, the reactor is a region where themicrofluidic channel or tube passes through that causes a reaction. Theregion may comprise various temperature zones. For example, forpolymerase chain reaction (PCR), the region comprises temperature zonesat specific temperatures comprising tube wraps between the temperaturezones. In some instances, the region comprises at least or about 1, 2,3, 4, 5, 6, 7, 8, or more than 8 temperature zones. In some instances,the region comprises about 3 temperature zones. The temperature zonesmay allow for a hot start reaction, thermal cycling, annealing,extension, polymerization, or protein denaturation. In some instances,one or more cycles are performed in each temperature zone. In someinstances, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, or more than 80 cycles are performed. In someinstances, about 30 to about 40 cycles are performed. In some instances,the region comprises a temperature zone for reverse transcription. Insome instances, the region comprises a region where light impinges onthe microfluidic channel or tube to initiate a photo-reaction. In someinstances, the region comprises a region for focusing acoustic energy.

Following a reaction such as an amplification reaction, droplets mayflow to a detector. In some instances, a detector detects droplet size.In some instances, a detector is used to quantify nucleic acids orprotein. In some instances, a detector is used to quantify a product orproducts of a reaction or set of reactions in the droplet. In someinstances, the quantity of product or products of the reaction or set ofreactions can be correlated to a physical or chemical property of aspecies comprising the droplet.

C. Detector

Provided herein are systems and methods for detection of one or moredetectable properties of partitions of at least one dispersed phase in acontinuous phase, moving in serial flow through a conduit. Detectionsystems, also referred to as detectors herein, can include one or moreof a system for separating partitions prior to detection, a narroweddetection channel through which partitions flow for detection, anoptical restriction for restricting the amount of electromagneticradiation that reaches a photodetector (restricting the field of view ofthe photodetector), and/or lock-in amplification. Systems and methodsmay further include a plurality of coplanar or nearly coplanarphoto-detectors, use of one or more silicon photomultipliers, use of aconduit comprising a tube for detection, and/or other aspects asdescribed herein.

The simplest embodiment of a detector includes a conduit through whichpartitions flow in single file, e.g., in an emulsion of partitions ofdispersed phase in continuous phase, where the conduit includes aninterrogation region (also referred to as an interrogation space oroptical stage herein) at which detection occurs, and a detection elementfor detecting a signal from the emulsion, e.g., from a partition, as itflows through the interrogation region. In a fluorescence system, thedetector also includes one or more excitation sources to provideelectromagnetic radiation, e.g., light, to the interrogation region.Further possible components of detectors are described below.

In general, detectors can be used in any suitable serial flow system. Incertain embodiments, systems and method provided herein provide aprocess system fluidly connected to a detector, such as a detector asdescribed herein; for example, a detector that has at least one, two,three, four, five, or all of an optical restriction, separation systemfor separating partitions to be detected (increasing the averagedistance between partitions), and/or narrowed conduit at theinterrogation region where the cross-sectional area of the conduit isless than or equal to the average equivalent spherical cross-section ofthe partitions (such as less than or equal to 100%, 95%, 90%, 80%, 70%,60%, 50%, 40%, 30%, 20%, or 10% of the average equivalent sphericalcross-section of the partitions); and/or a detector that utilizeslock-in amplification, e.g., for the detection of multiple signals froma single partition and/or to improve signal-to-noise ration of one ormore signals from a single partition; and/or a detector that utilizes aconduit such as a tube at the interrogation region; and/or a detectorthat utilizes a coplanar or nearly coplanar array of a plurality ofexcitation sources and/or one or more detection elements, such as adetector where the conduit in the interrogation region is a tube orsimilar structure; and/or a detector where all or substantially all ofthe surfaces of parts of the detector that contact dispersed phasepartitions have a greater affinity for continuous phase than fordispersed phase, for example, at least 90, 95, 99, or 99.9% of surfaces.The process system can be any suitable process system, for example asystem that includes a partitioner for generating partitions, e.g., froma sample, such as any of the partitioners described herein; or a reactorfor generating and/or modulating a reaction in at least a portion ofpartitions, such as any of the reactors described herein, e.g., athermal cycler such as a thermal cycler for PCR, e.g., thermal cyclersas described herein, or a combination thereof.

In certain embodiments, systems and method provided herein provide aprocess system and a intake system, where the process system is fluidlyconnected to a detector, such as a detector as described herein, andwhere the intake system provides one or more samples to the processsystem, where the intake system and the process system are never incontinuous fluid communication, such as any of the intake and processsystems described herein, for example a intake system and a processsystem that are joined by an injector, where, e.g., the injector cancycle between connection to the intake system and connection to theprocess system, but does not provide a continuous connection between theintake system and the process system. The detector can be any suitabledetector, such as a detector as described herein; for example, adetector that has at least one, two, three, four, five or all of anoptical restriction, separation system for separating partitions(increasing the average distance between partitions) to be detected,and/or narrowed conduit at the interrogation region where thecross-sectional area of the conduit is less than or equal to the averageequivalent spherical cross-section of the partitions; and/or a detectorthat utilizes lock-in amplification; and/or a detector that utilizes aconduit such as a tube at the optical stage; and/or a detector thatutilizes a coplanar or nearly coplanar array of a plurality ofexcitation sources and/or one or more detection elements, such as adetector where the conduit in the interrogation region is a tube orsimilar structure; and/or a detector where all or substantially all ofthe surfaces of parts of the detector that contact dispersed phasepartitions have a greater affinity for continuous phase than fordispersed phase.

Partitions moving through the conduit may be any size, e.g., volume, asdescribed herein, such as 0.01-100 nL, 0.05-50 nL, 0.1-10 nL, 0.1-5 nL,0.1-3 nL, or 0.1-2 nL, or any other suitable volume as describedelsewhere herein. A series of partitions moves through the conduit insingle file. In certain embodiments, the partitions arrive at thedetector in groups, e.g., a group of partitions derived from a singlesample; in some cases the groups of partitions are separated from eachother by, e.g., spacer fluid; in certain embodiments, the spacer fluid,one or more partitions, or both, have one or more properties, such asone or more optical properties, that can be used to delineate boundariesbetween groups of partitions. A group of partitions can comprise anysuitable number of individual partitions, for example, 10-10,000,000,10,000-2,000,000, 100-1,000,000, 200-500,000, 500-500,000, 1000-200,000,5000-200,000, 10,000-200,000, 10,000-40,000, 20,000-200,000,25,000-45,000, or any number as described herein.

Systems and methods provided herein can allow for detection of a singlepartition at a time, with little or no signal overlap from adjacentpartitions, for example, where signal that reaches a detection elementfrom the interrogation region that is from a partition is at least 60,70, 80, 90, 95, 99, 99.5, or 99.9% from a single partition in theinterrogation region.

In certain embodiments, such as in digital assays (e.g., digital PCR),partitions are counted as either positive (containing a component ofinterest, for example, a nucleic acid that has been amplified) ornegative (not containing a component of interest, for example, notcontaining a nucleic acid that has been amplified). In certainembodiments, at least 60, 70, 80, 90, 92, 95, 96, 97, 98, 99, 99.5, or99.9% of the partitions flowing through the detector, for example,partitions in a group of partitions such as a group of partitions thatcorresponds to a sample, can be detected as individual partitions, e.g.with sufficient resolution to be counted as positive or negative. Thisresolution can be achieved by aspects of the system and methods asdescribed herein, such as a detector as described herein; for example, adetector that has one, two, three, four, five, or all of an opticalrestriction, separation system for separating partitions to be detected(increasing average distance between partitions), and/or narrowedconduit at the interrogation region where the cross-sectional area ofthe conduit is less than or equal to the average equivalent sphericalcross-section of the partitions; and/or a detector that utilizes lock-inamplification, such as in a detector with a plurality of excitationsources and at least one detection element; and/or a detector thatutilizes a conduit such as a tube at the interrogation region; and/or adetector that utilizes a coplanar or nearly coplanar array of aplurality of excitation sources and/or one or more detection elements;and/or a detector where all or substantially all of the surfaces ofparts of the detector that contact dispersed phase partitions have agreater affinity for continuous phase than for dispersed phase.

Systems and methods provided herein also can allow for determination ofthe volume of individual partitions as they flow through the detector;this, together with the number of partitions, can, e.g., allowcalculation of an overall volume for, e.g., a group of partitions, suchas a group of partitions that corresponds to an individual sample. This,together with other information, such as the number of partitions thatgive a positive signal in the group for a particular marker of aparticular component, can allow accurate determination of the initialconcentration of one or more components in the original sample fromwhich the partitions were derived.

For convenience, detection systems will be described in terms offluorescence systems, i.e., a system where a fluorophore is excited byelectromagnetic radiation at one range of wavelengths and emitselectromagnetic radiation at a second range of wavelengths, which isdetected. However, any suitable method of detection may be used, and itwill be appreciated that many aspects of the systems and methodsdescribed herein are applicable to a wide range of types of detection,e.g., chemiluminescence, radiation, absorbance, scattering, Ramanscattering, electrical capacitance, electrical current, electricalresistance, thermal mass, image capture, and the like.

Detection systems and methods described herein are generally applicableto detection of signal from partitions moving in serial flow through aconduit. The source of the partitions may be any suitable source; ingeneral, partition detection will be described in terms of detectionfollowing reaction, e.g., thermal cycling for PCR, but it will beunderstood that any suitable droplet source that produces partitions, atleast some of which have or may have detectable properties, may be used.

It can be desirable to detect one or more signals from a singlepartition at a time, with little or no signal from adjacent partitionsbeing detected, as the partitions flow through a conduit. Systems andmethods provided herein can allow for detection of a single partition ata time, with little or no signal overlap from adjacent partitions, forexample, where signal that reaches a detection element from theinterrogation region that is from a partition is at least 60, 70, 80,90, 95, 99, 99.5, or 99.9% from a single partition in the interrogationregion. Systems and methods provided herein can include one or more, forexample two or more, such as all three, of 1) increasing separation ofpartitions prior to or during detection (increasing average distancebetween partitions); 2) narrowing of the conduit at the interrogationregion; and/or 3) an optical restriction to restrict the amount ofelectromagnetic radiation reaching a detection element, such as aphotodetector. Using one or more of these systems and methods, it can bepossible to detect signal from separate partitions, so that signaldetected from an individual partition is at least 80, 90, 95, 96, 97 98,99, 99.5, or 99.9% and/or not more than 90, 95, 96, 97 98, 99, 99.5,99.9, or 100% due to signal produced by that partition, and not fromadjacent partitions.

In certain embodiments, of the systems and methods provided herein, asystem for increasing the average distance, i.e., separation, betweenpartitions of dispersed phase in a continuous phase is used prior toand/or concurrent with partitions reaching an interrogation region inwhich detection of partitions occurs. Partition separation systems canincrease separation of partitions (the average distance betweenpartitions) by any suitable operation, for example, by adding a secondcontinuous phase to a flow of partitions of dispersed phase in a firstcontinuous phase (where the second continuous phase can be the same asor different from the first continuous phase), by narrowing the conduitthrough which partitions flow in continuous phase, or a combinationthereof. Separation systems increase the average distance (separation)‘a’ between partitions of dispersed phase in the continuous phases to avalue ‘b’, where b>a. “a” and “b” can be, e.g., the distance between thegeometric centers of adjacent partitions, or other suitable measuringpoint. In certain embodiments, systems and methods provided hereinincrease separation of partitions (average distance between partitions)so that b is at least 102, 105, 110, 125, 150, 175, 200, 225, or 300% ofa, and/or b is at most 105, 110, 125, 150, 175, 200, 225, 300 or 400% ofa, for example, b can be 102-400%, such as 102-300%, or in some cases102-200% of a. Alternatively or additionally, separation of partitionscan be described in terms of average distance from the surface of onepartition to the surface of an adjacent partition. Thus, partitions canbe separated prior to reaching the interrogation region so that theaverage distance from the surface of one partition to the surface of anadjacent partition is 20-500%, 20-400%, 30-300%, 50-200%, 75-200%,50-150%, or 75-125% of the average spherical diameter of the partitions.Partitions can be separated prior to reaching the interrogation regionso that the average distance from the surface of one partition to thesurface of an adjacent partition is 20-500 um, or 20-400 um, or 30-300um, or 50-200 um, or 75-200 um, or 50-150 um, or 75-125 um.

In certain embodiments, a second continuous phase is added to the flowof partitions of dispersed phase in a first continuous phase, prior tothe partitions reaching the interrogation region, for example, justprior to droplets reaching an interrogation region. The composition ofthe first and second continuous phases can be the same or different. Incertain embodiments, continuous phase is removed from the flow ofpartitions after they have passed through the interrogation region, andpart or all of the removed continuous phase is reintroduced into thesystem, e.g., some or all of the removed continuous phase can be used asthe second continuous phase in a separation system. Alternatively oradditionally, continuous phase removed from the flow of partitions afterthe partitioner can serve as a source of second continuous phase forseparation of partitions (increasing average distance betweenpartitions). See, e.g., Disengagement, discussed for partitioners, forfurther discussion of removal and reintroduction of continuous phase.Sufficient second continuous phase may be added to the flow ofpartitions in the first continuous phase that the total continuous phasevolume in the flow pathway is increased by a suitable amount to achievea desirable separation of droplets, e.g., an increase of at least 2, 5,7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,or 95% and/or not more than 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 100, 120, 150, 170, 200, 250, or 300%,such as between 10-300%, for example 10-150%, in some cases 15-125%.Sufficient second continuous phase can be added to the flow pathway sothat the average distance between partitions (e.g., as measured from thesurface of one partition to the surface of an adjacent partition) is atleast 1, 2, 5, 10, 30, 50, 70, 80, 90, 100, 110, 120, 130, 150, 170,200, 250, or 300 um and/or not more than 2, 5, 10, 30, 50, 70, 80, 90,100, 110, 120, 130, 150, 170, 200, 250, 300, 400, or 500 um, such as30-300 um, or 50-250 um, or 50-150 um. Where the separation betweenpartitions (e.g., as measured from the surface of one partition to thesurface of an adjacent partition) is expressed in terms of the averagespherical diameter of partitions, it can be, e.g. at least 10, 20, 50,70, 80, 90, 100, 110, 120, 150, or 200% and/or not more than 20, 50, 70,80, 90, 100, 110, 120, 150, 200, 300, or 500% of the average sphericaldiameter of partitions.

In certain embodiments, partitions are detected in an interrogationregion of conduit, where the cross-sectional area of the interrogationregion is equal to or less than the average spherical cross-sectionalarea of partitions of dispersed phase that pass through theinterrogation region. Any suitable reduction in cross-sectional area maybe used. In certain embodiments, the cross-sectional area of the conduitmay be not more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%and/or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% and/or atleast 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% and/orat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, of the equivalentaverage spherical cross-sectional area of the partitions of thedispersed phase. For example, the cross-sectional area of the conduit inthe interrogation region may be 10-100%, such as 20-100%, for example40-60% of the equivalent average spherical cross-sectional area ofpartitions of the dispersed phase. The conduit prior to theinterrogation region may be, e.g., 100-400 um in diameter (if circularcross-section) and the conduit in the interrogation region may be, e.g.,20-120 um in diameter (if circular cross-section).

In certain embodiments of the systems and methods provided herein, anoptical restriction is placed in the path of electromagnetic radiationemitted from partitions in an interrogation region, so that only afraction of the electromagnetic radiation reaches a detection element,e.g., photodetector, than could otherwise reach it, i.e., without therestriction. The optical restriction can be configured and positioned sothat electromagnetic radiation emanating from partitions upstream anddownstream of the partition in the interrogation region is reduced inits ability to reach the detection element, i.e., blocked; thus, theoptical restriction will generally be positioned at a point that is ator near the middle of the interrogation region, and have a width orother suitable dimension that reduces or eliminates electromagneticradiation from sources other than the partition in the interrogationregion, given the likely spacing of partitions in the flow, volume ofthe partitions, and cross-sectional area of the conduit in theinterrogation region. In certain embodiments, the optical restrictionhas a configuration and position that reduces electromagnetic radiationreaching the detection element to not more than 80, 50, 40, 30, 20, 10,5, 2, 1, 0.1, 0.01, 0.001, 0.0001, or 0.00001%, and/or at least 50, 40,30, 20, 10, 5, 2, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, or 0.000001% ofthe electromagnetic radiation that would reach the detection elementwithout the optical restriction, e.g., under standardized conditionssuch as excitation of fluorophores in a partition that are present at aconcentration that indicates, e.g., the presence and amplification of asingle nucleic acid in the partition; in certain embodiments, the amountof electromagnetic radiation reaching the detection element is0.000001-5% of the electromagnetic radiation that would otherwise reachthe detection element. Other suitable methods of standardization will beapparent, based on the type and extent of the process that thepartitions are likely to undergo in the system; in general,standardization is based on an “average” partition that contains acomponent that would, e.g., render the partition as a positive signal inthe system. In the example above, this would be a partition in a PCRsystem that contained 1 nucleic acid that was amplified in the PCRreaction. In certain embodiments, the optical restriction is configuredand positioned so that, either with the optical restriction alone or incombination with one or both of separation of partitions (increasing theaverage distance between partitions) and/or narrowing of theinterrogation region, at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95%, 97%, 98%, or 99% and/or not more than 30%, 40%, 50%, 60%, 70%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% of the electromagnetic radiationreaching the detection element was emitted by at least one component ina single partition of the dispersed phase, such as 60-100%, or 80-100%,or 90-100%, or 95-100%, or 98-100%, or 99-100%, or 99.5-100%, or99.9-100% was emitted by a single partition. In certain embodiments, theoptical restriction is configured and positioned so that, either withthe optical restriction alone or in combination with one or both ofseparation of partitions and/or narrowing of the interrogation region,when a first partition is passing through the interrogation region, notmore than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%,40%, or 50% and/or at least 0, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, or 40% of theelectromagnetic radiation reaching the detection element due to one ormore components in a partition is from one or more partitions other thanthe partition in the interrogation zone, such as not more than 50%, forexample, not more than 30%, such as not more than 25%. Any suitableshape may be used for the optical restriction, e.g., a circle (pinhole),a slit, two slits (square), and other shapes as described herein. Theoptical restriction can be placed at any suitable distance from theinterrogation region, e.g., 0.1-24 inches, or 1-5 inches, or 2-4 inches,such as about 3 inches. The optical restriction can be placed at anysuitable distance from the surface of the detection element, e.g.,0.1-24 inches, or 0.1-5 inches, or 0.1-2 inches, or 0.1-0.5 inches,e.g., about 0.25 inches. The area of the optical restriction will bedetermined by its placement and by the desired proportion of light toreach the detection element, such as, for a pinhole (circle), an areacorresponding to a diameter of 10-1000 um, such as 100-250 um; forexample, in an embodiment where the optical restriction is a pinhole andis positioned 3 inches from the interrogation region and 0.25 inchesfrom the detection element, the diameter of the optical restriction is200 um. The thickness of material comprising the optical restriction ispreferably thin, e.g., as thin as possible.

In certain embodiments, lock-in amplification is used as part of thedetector systems and methods. Lock-in amplification provides a number ofadvantages, as detailed herein; for example, lock-in amplification canallow the separate measurements of signals emitted from a singlepartition dud to electromagnetic radiation from a plurality ofexcitation sources with only a single detection element. In lock-inamplification, a signal source (e.g. an excitation source) is modulatedby a carrier periodic function. The resulting signal is multiplied bythe carrier periodic function and integrated over multiple periods ofthe carrier periodic function. Because background noise, whentransformed into frequency space, will typically contain components at awide range of frequencies, use of this method allows for acceptance ofonly that noise in a small bandwidth around the frequencies of greatestrelative magnitude in the Fourier series representative of the carrierperiodic function (related to the integration time), while the signal isconcentrated within this frequency range or set of frequency ranges. Asa result, noise outside of this bandwidth is rejected, and the relativelevel of signal to the level of noise is greatly amplified. In someinstances, signal-to-noise can be improved by more than six orders ofmagnitude.

As applied to the present systems and methods, lock-in amplification canbe practiced by modulating each excitation source simultaneously at aset of unique frequencies. Exemplary methods include use of an opticalchopper in front of the excitation source where there is a physicalobject that blocks then passes electromagnetic radiation at a prescribedfrequency based on the size of the feature and the rate of spinning; orLED blinking by fast on/off current of the system, or any other suitablemethod.

In certain embodiments, a single photodetector is used and the periodicfunctions are sunusoidal. Because noise in these systems is concentratedin lower frequency bands (e.g. 1/f noise, power supply noise, mainsvoltage noise), it is desirable that the modulation frequency be at amuch higher frequency than the portion of the spectrum with the mostnoise. In some cases, this will be at frequencies greater than 100 kHz.In other cases, this will be at frequencies greater than 1 MHz. For eachchannel, lock-in detection can be achieved by multiplying the resultingsignal at the photodetector by that channel's modulation frequency andintegrating over at least one period of the modulation frequency. Thismathematical operation can be achieved by analog means (e.g. using asignal multiplier circuit followed by a low pass filter) or with digitalalgorithms (e.g. using analog-to-digital converters to digitize thesignal, followed by either numerical integration, numerical filteralgorithms, or Fourier transform/Fast-Fourier Transform operations andfiltering). By choosing a small enough low-pass filter bandwidth (e.g. 2kHz), only noise contained in a bandwidth of that size centered aroundthe modulation frequency in the original signal will be retained. Thisallows for significant increases in the signal-to-noise ratio. Moreover,if the individual excitation sources are modulated at sufficientlyspaced intervals (e.g. 1 MHz, 1.1 MHz, 1.23 MHz, and 1.32 MHz for a fourchannel system with a bandwidth of 1 kHz), when demodulating the signalfor one of the modulation frequencies, the remaining modulationfrequencies are rejected with the noise. By demodulating these signalsin parallel, any number of channels may be multiplexed on a singlephotodetector simply by adding additional excitation sources modulatedat unique frequencies.

Thus, in certain embodiments, lock-in amplification is used. This hasthe advantages of the simplicity of the optical arrangement, theimproved signal-to-noise ratio (potentially allowing for the use of alower cost photodetector), and the requirement for only a singlephotodetector. Further details on lock-in amplification are given in thedescription of FIG. 65. In certain embodiments, at least 2, 3, 4, 5, 6,7, or 8 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15excitation sources are used, for example, 2-10, such as 2-7, forexample, 2-6 excitation sources with a single detection element, e.g.,photodetector, to detect electromagnetic radiation emitted in responseto all of the excitation sources, where lock-in amplification is used.

In certain embodiments, an interrogation region is used where theinterrogation region comprises a conduit whose walls have the same orsubstantially the same transmittance for one or more wavelengths ofelectromagnetic radiation of interest, for example, one or moreexcitation wavelengths and one or more emission wavelengths, around thecircumference of the conduit. In certain embodiments, the conduit is atube, for example a tube with a circular or substantially circularcross-section. The conduit can comprise any suitable material, forexample, a material that has higher affinity for the continuous phasethan the dispersed phase; in embodiments, the conduit comprisefluoropolymer, e.g., for use with a fluorinated oil continuous phase.

Use of such a conduit, e.g., a tube, at the interrogation region meansthat a plurality of excitation sources, a plurality of detectionelements, or both, can be arranged so that all of the excitation sourcesand/or detection elements are in the same plane or nearly in the sameplane, where the plane is orthogonal to the long axis of the conduit asit passes through the interrogation region. For example, all of theexcitation sources and/or detection elements can be within 50, 40, 30,20, 15, 10, 5, 4, 3, 2, 1, 0.5, or 0.1 degree of a plane orthogonal tothe long axis of the conduit, i.e., orthogonal to the direction of flowof partitions in the conduit. In certain embodiments, at least 2, 3, 4,5, 6, 7, or 8 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15excitation sources are used, for example, 2-10, such as 2-7, forexample, 2-6 excitation sources, all of which are within 50, 40, 30, 20,15, 10, 5, 4, 3, 2, 1, 0.5, or 0.1 degree of a plane orthogonal to thelong axis of the conduit; in certain embodiments, in addition to aplurality of excitation sources, at least one detection element is alsowithin 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, or 0.1 degree of theplane orthogonal to the long axis of the conduit in which the excitationsources are situated.

In certain embodiments, surfaces of the conduit through which thepartitions pass in the detector have a greater affinity for continuousphase than for dispersed phase. For example, the surfaces may befluoropolymer when the continuous phase is, e.g., a fluorinated oil.

Thus, described herein are various systems and methods for detectingindividual droplets (partitions). An individual droplet (partition) maybe detected based on the fluorescence of the individual droplet(partition). In some instances, as the individual droplet (partition)enters the optical stage (interrogation region), a signal is detectedthat is an increased over baseline. In some instances, an individualdroplet (partition) is distinguished by peaks in the optical signal. Insome instances, fluorescent molecules are added to the dispersed phaseprior to droplet (partition) generation to increase the optical signalover the baseline optical signal when droplets (partitions) are not inthe center of the optical stage (interrogation region). In someinstances, the fluorescent molecules are added to the dispersed phaseswithout quenching molecules. In some instances, the fluorescentmolecules comprise a similar excitation wavelength and/or emissionwavelength as the fluorophores for the assay or reaction. In someinstances, the fluorescent molecule comprises a different excitationwavelength and/or emission wavelength as the fluorophores for the assayor reaction.

In some instances, an arrangement for a detector detects a singledroplet (partition) at a time. An excitation source may transmitelectromagnetic radiation (e.g., light) in substantially a singledirection. The light may pass through a filter and then passes through alens. The lens may focus the light onto an optical stage. Droplets(partitions) may pass through the optical stage. Light may be emitted bythe fluorescent molecules in the optical stage. In some instances, adetection lens, filter, optical restriction, or combinations thereof areplaced substantially in line with the excitation source and on anopposite side of the optical stage. In some instances, a detector isplaced beyond an optical restriction. Light may pass from the excitationsource and may be limited to a single wavelength by the filter. Thelight may be focused by the lens on a droplet (partition) where emissionoccurs. The emission may be then collimated by the second lens so thatan image of the optical stage may be produced on the plane of theoptical restriction. The optical restriction may select only a portionof the light corresponding to a region of the optical stage thatcomprises the droplet (partition). The detector may then register asignal for the light. In some instances, the detector is substantiallyorthogonal to both the excitation source and the direction of droplettravel. Exemplary excitation sources include, but are not limited to, aLED, a laser, or any other light source substantially restricted to asmall range of wavelengths. The detector may be a photodiode, PMT, SiPM,or any other optical detector where the signal level increasesmonotonically with light intensity. In some instances, the excitationsource is mounted in a light tube. In some instances, an optical fiberwith integral lens is used to position the excitation source over theoptical stage. In some instances, the excitation filter is chosen sothat only light near the excitation frequency passes the filter. In someinstances, the emission filter is chosen so that only light near theemission frequency passes the filter.

In some instances, an arrangement comprises both emission and excitationlight that travel on substantially the same path from an optical stage.The system may comprise an excitation source, an excitation filter, adichroic mirror, an optical stage lens, an optical stage, a detectionfilter, a detection lens, a detection optical restriction, and adetector. The excitation source may emit light at substantially a singlewavelength which is restricted to primarily that wavelength by theexcitation filter. The light may strike the dichroic mirror, where it isturned approximately 90 degrees toward the optical stage lens, whichfocuses the light on the optical stage. The light may excite fluorescentmolecules in droplets (partitions) in the optical stage, and lightemitted by those molecules passes back through the optical stage lens,where it is collimated onto the dichroic mirror. The light may passthrough the dichroic mirror, through the emission filter which restrictsthe light primarily to the wavelength emitted by the fluorescentmolecules in the droplets (partitions), where it then passes through thedetection lens and is focused on the plane containing the opticalrestriction. The optical restriction may restrict the light passing tothe detector to only that portion corresponding to a single droplet(partition) in the optical stage. The light creates a signal on thedetector that monotonically increases with the emission intensity fromthe fluorescent molecules in a single droplet (partition). In someinstances, the further comprises a mirror behind the optical stage so asto collect more emitted light from the droplets (partitions) in theoptical stage.

The detector is what senses the presence and/or level of detectablecomponents in the partitions (here described in terms of fluorophores).Thus, for fluorescence systems, the detector is what senses the presenceand/or level of fluorophores in the partitions and, e.g., allowsdiscrimination between “positive” (e.g., contains a molecule ofinterest) and “negative” (e.g., does not contain a molecule of interest)partitions in digital assays. The underlying concentration of thatmolecule in the original sample can then be determined from Poissonstatistics on the ensemble of partitions.

One basic principle of detection in fluorescence systems is excitationof a fluorophore in the partition, which then emits electromagneticradiation that can be measured with a photodetector. The intensity ofthe electromagnetic radiation emitted for a given excitation intensityis proportional to the concentration and state of the fluorophore in thepartition. Fluorophores may be associated with molecules of interest inany suitable manner. Fluorophores can be incorporated into largermolecular systems such that the intensity of emission for a givenexcitation intensity can be related to the concentration of anotherchemical entity in the partition. For example, if a nucleic acid bindingdye (e.g. intercalating or bis-intercalating agents, minor or majorgroove binders, or external binders) is used (e.g. SYBR Green,EvaGreen), the intensity of emission can be related to the total amountof nucleic acid in the system in which the intercalating dye isintercalated. For a hydrolysis probe, in which a fluorophore is closelyassociated with a quencher in the unhydrolyzed state but is remote fromthe quencher in the hydrolyzed state, the emitted intensity can beassociated with the concentration of dye released from the hydrolyzedprobe; hydrolysis can occur during nucleic acid polymerization if theprobe incorporates a oligonucleotide sequence complementary to thesequence of nucleic acid of interest when using a polymerase with 5′->3′exonuclease activity. Alternatively the addition of fluorescentdye-modified nucleotides (e.g. dye systems with tetra- or pentaphosphatelinked fluorophores) may produce a fluorescent signal with intensityproportional to the total amount of nucleic acid polymerization. Theratio of unmodified nucleotides to modified nucleotides tunes the totalpotential intensity achievable in each reaction vessel.

Excitation sources can generally be anything that emits electromagneticradiation at wavelength ranges where the fluorophores can absorb thisenergy to re-emit. Noise can arise in these systems due to backgroundelectromagnetic radiation from the excitation source as well asauto-fluorescence. Thus, it is generally preferable that theelectromagnetic radiation that reaches the partitions be restricted tothe wavelengths that will excite the fluorophores as much as practicallypossible. This can be done in any suitable manner, for example, by (1)using electromagnetic radiation sources that emit electromagneticradiation primarily at the wavelengths that will excite the fluorophoresof interest, (2) using electromagnetic radiation sources that primarilyemit electromagnetic radiation in the direction of the partitioncontaining the fluorophores of interest, (3) using refractory elementsand/or reflective surfaces to concentrate the electromagnetic radiationonto the interrogation region, and/or (4) using optical filters torestrict the wavelengths of electromagnetic radiation reaching thepartitions containing the fluorophores of interest, or any combinationof these methods. For (1), examples of electromagnetic radiation sourcesare light-emitting diodes (LEDs); lasers, including but not limited toHelium Neon, Argon Ion, Krypton Ion, Xenon Ion, Carbon Dioxide,Neodymium Doped Crystal Yttrium, Aluminum Garnet, Titanium Sapphire ModeLocked, Excimer, Semiconductor Diode, or dye-based; excited phosphors;excited quantum dots; or arc discharge lamps. For (2), suitableelectromagnetic radiation sources are lasers, which concentrate theirenergy into a small beam or cone rather than emitting hemispherically.Other options for (2) can be collimated light emitting diodes (LEDs),excited phosphors, excited quantum dots, or arc discharge lamps. For(3), refractory elements include lenses; any suitable lens may be used,such as achromats/asphericals, and the like, including but not limitedto semi-apochromats, apochromats, meniscus, hemispherical, cone, rod,ball, and cylinder lenses. Reflective elements can include parabolicmirrors, dichroic mirrors, or any other suitable reflective element,including but not limited to prisms and beam splitters. For (4), filtersare preferentially bandpass filters, passing only a narrow range offrequencies substantially near the frequencies of electromagneticradiation emission by the excitation source. Short-pass, long-pass,notch, neutral density or polarizing filters may be used; dichroicmirrors may also be used.

Photodetectors provide signals that change monotonically with theintensity incident or power of electromagnetic radiation on theirsurfaces. The choice of photodetector in the detection system depends onthe incident intensity or power. For arrangements with high incidentintensity, relatively insensitive detectors (e.g. photodiodes) may beused, which has the advantage of low cost. For other arrangements withintermediate intensity, charge coupled device (CCD) detectors or siliconphotomultipliers may be used. For arrangements with low incidentintensity, very sensitive devices (e.g. photomultiplier tubes or siliconphotomultipliers) will typically be used to improve signal-to-noiseratio. In certain embodiments, one or more silicon photomultipliers isused.

In some situations, it is desirable to detect and/or measure a quantityof more than one component in each partition (“multiplexing”). This canachieved by any suitable technique, for example: (1) associating acomponent to each fluorophore, exciting each fluorophore separately,detecting the emitted intensity from each fluorophore, and using theintensity to calculate a quantity for the component associated with eachfluorophore; (2) associating all but one of the components to eachfluorophore and incorporating a fluorophore that is proportional to thetotal amount of all of the components in the system, exciting eachfluorophore separately, detecting the emitted intensity from eachfluorophore, using the intensity to calculate a quantity for thecomponent associated with each fluorophore, using the intensity from thenon-specific fluorophore to calculate a quantity for the entire mixture,and using the total quantity and the quantity of each component otherthan the final component to determine the quantity of the finalcomponent; (3) associating two or more components to each fluorophoreeach of which generates a specific fluorescent intensity, exciting eachfluorophore separately, detecting the emitted intensity from eachfluorophore, and using the intensity to detect the quantity for eachcomponent associated with each fluorophore at each intensity level; (4)associating a component to each fluorophore, exciting each fluorophoresimultaneously, detecting the emitted intensity from each fluorophore,and using the intensity to calculate a quantity for the componentassociated with each fluorophore; (5) associating all but one of thecomponents to each fluorophore and incorporating a fluorophore that isproportional to the total amount of all of the components in the system,exciting each fluorophore simultaneously, detecting the emittedintensity from each fluorophore, using the intensity to calculate aquantity for the component associated with each fluorophore, using theintensity from the non-specific fluorophore to calculate a quantity forthe entire mixture, and using the total quantity and the quantity ofeach component other than the final component to determine the quantityof the final component; (6) associating two or more components to eachfluorophore each of which generates a specific fluorescent intensity,exciting each fluorophore simultaneously, detecting the emittedintensity from each fluorophore, and using the intensity to detect thequantity for each component associated with each fluorophore at eachintensity level.

Signals from different fluorophores can be discriminated in any suitablemanner. In a first method, a non-spectrally sensitive photodetectionsystem may be used and the excitation source at any given time may belimited to excitation wavelengths that primarily excite only onefluorophore in the system. While the excitation bands for desiredfluorophores may overlap and thus more than one fluorophore may beexcited in this case, corrections to the detected intensity on thephotodetector may be made from a set of standard calibrations. Bycycling through the excitation sources, each of the fluorophores may bedetected at the photodetector, and, by correlating the time that eachexcitation source was on with those measurements at the photodetector, aquantity may be calculated for each component (or group of components)associated with that fluorophore. Each excitation source may beactivated immediately after de-activating the previous activationsource, or there may be a period where no activation source is on. Eachexcitation source should be active for at least the amount of timerequired for the signal from its associated fluorophore to stabilize. Asingle data sample may be taken from the photodetector during this time.Alternatively, multiple data signals may be taken from the photodetectorduring this time and aggregated. An advantage of this approach is thatit is very optically simple and only requires a single photodetector.Disadvantages are that the gain will typically be uniform across allwavelengths and the number of samples at each wavelength for a givensampling frequency will be smaller than they would be were all thewavelengths of interest excited simultaneously.

A second method for discriminating signals from different fluorophoresis to excite one or more fluorophores simultaneously, spatially separatethe electromagnetic radiation emitted from the fluorophores, and detectall or a subset of the separated electromagnetic radiation with one ormore photodetection elements spatially distinct from each other(“spectrometer method”). Means of spatially separating electromagneticradiation include resolving the emitted electromagnetic radiation bywavelength, e.g., with a diffraction grating (transmissive orreflective), mirrors, a refractive prism, a series of dichroic mirrorsthat selectively remove one wavelength range at a time, or othersuitable means. The refracted electromagnetic radiation can be collectedby one or more photodetectors. In an example, the photodetectors areeither photodiodes or silicon photomultipliers that are spatiallydistinct from each other and are positioned so as to intercept thewavelengths primarily associated with each fluorophore. In anotherinstance, a linear charge-coupled device (CCD) or complementarymetal-oxide-semiconductor (CMOS) sensor is used as the photodetector,and it is positioned to intercept electromagnetic radiation across thewavelength range emitted by some or all of the fluorophores. In someinstances, it may be desirable to minimize the size of thephotodetection elements (e.g. for cost or compactness), and an opticalconcentrating element may be used (e.g. lens, focusing mirror) toconcentrate a spatial band of electromagnetic radiation onto eachphotodetection element.

A third method of discriminating signals from different fluorophoreswhile simultaneously improving signal-to-noise ratio is to use lock-inamplification. See description above and below for lock-inamplification.

Thus, detectors for use in systems and methods as described herein mayallow for multiplexing. In some instances, multiple wavelengths are usedto detect separate emission spectra by using separate signal channels.In some instances, the signals received by the signal channels areseparated in time. In some instances, the signal channels are generatedby different detectors. The separate channels may allow for measuringdifferent levels of intensity of each wavelength. In some instances, thesignal of each channel varies temporally or spatially.

Detectors for use in systems and methods as described herein maycomprise multiple channels. In some instances, one channel is used fordetecting droplet size. In some instances, the channel for detectingdroplet size comprises a fluorophore having an intensity that does notvary with the state of one or more reactions in the droplet. In someinstances, detectors further comprise one or more channels for measuringa chemical reaction. Such channels may have an emission intensity thatdepends on the state of the chemical or physical reactions that comprisethe underlying assays. In some instances, use of one or more channelsindicates whether a droplet (partition) is present or not. For example,if a portion of the one or more channels are above a threshold, thedroplet (partition) is present. In some instances, if a portion of theone or more channels are below a threshold, the droplet (partition) isnot present.

Droplets (partitions) may be detected in a system comprising multiplechannels. In some instances, size of the droplet (partition) isdetermined by measuring a time from a signal from the channel to riseabove a threshold to a time for a signal from the channel to fall belowa threshold. In some instances, size of the partition is determined bymeasuring the width, in time, of a signal peak at a fraction of themaximum signal for that peak. In further instances, the fraction isbetween 0.25 and 0.35, 0.45, and 0.55, 0.2 and 0.8, 0.65 and 0.85, and0.1 and 0.9. In some instances, one or more measurements are detected.In some instances, the one or more measurements improve resolution ofthe size. In some instances, at least or about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 28, 32, 36, 40, 44,50, 60, 70, 80, 100, 200, 1000 or more than 1000 measurements aredetected.

Thus, in certain embodiments detectors are configured for multiplexing.Exemplary diagrams of systems for achieving multiplexing are illustratedin FIG. 93 and FIG. 94. FIG. 93 illustrates temporal multiplexing. Insome instances, multiple excitation sources and/or a controller is used.In some instances, the excitation filters and/or emission filters areconverted to multiple bandpass filters. In some instances, the multiplebandpass filters substantially pass only those wavelengths correspondingto each of the excitation or emission wavelengths in the system. Thecontroller may control whether the excitation sources are on or off. Thesystem 1921 comprises multiple excitation sources 1901, 1903, anexcitation filter 1905, an optical stage 1907, an emission filter 1909,an optical restriction (e.g. a pinhole or slit) 1911, a photo-detector1913, and a controller 1915. Each of the excitation sources 1901, 1903produces light over a range of wavelengths, where the wavelength rangeof each of the excitation sources does not substantially overlap withthe wavelength range of any other of the excitation sources so that atleast one fluorescent molecule in the dispersed phase is excited by eachof the excitation sources 1901, 1903 and at least one fluorescentmolecule in the dispersed phase is not excited by that excitationsource. The controller 1915 is connected to the excitation sources 1901,1903 and controls whether any of the excitation sources 1901, 1903 is onat any given time. By coordinating the times when a given excitationsource is active with the timing of measurements made by thephoto-detector 1913, the emission from only a specific fluorophore canbe detected with a non-wavelength specific detector. Systems asdescribed herein can comprise a time scale over which the fluorescentmolecules react to changes in excitation, a time scale for changes inexcitation intensity, and a time scale for making measurements on thedetector that may be shorter than a time required for a droplet to moveacross the optical stage. The excitation sources may comprise LEDs,lasers, excited phosphorescent material, quantum dots, any otherelectromagnetic radiation (light) emitting material for which the lightis emitted in a narrow range, or any combination thereof.

In some instances, a first excitation source is turned on by thecontroller for a first time. During this time, at least one measurementis taken by the detection element. In some instances, one measurement istaken by the detector. In some instances, multiple measurements aretaken by the detector. For example, at least or about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12 or more than 12 measurements are taken by the detector.In some instances, at least 5 measurements are taken by the detector.These measurements may be averaged to get a noise-stabilizedmeasurement. In some instances, at least one of the measurements at thestart of the time when the first excitation source is on is rejected,and only the remaining measurements are aggregated. In some instances,no aggregation of the measurements is taken. In some instances, a medianmeasurement is taken. After a first period of time has elapsed, thefirst excitation source may be deactivated and a second excitationsource is activated. A second set of one or more measurements may betaken by the detector. In some instances, the one or more measurementsof the second set are aggregated. In some instances, the one or moremeasurements are not aggregated. After a second period of time haselapsed, the second excitation source may be deactivated. If there areadditional excitation sources, these may be activated and de-activatedin turn, with each comprising its own time period of activation. Afterall of the sources have been turned on, the controller may loop backaround to the first excitation source and the process repeats.Generally, the time period for each excitation source can all bedifferent. In some instances, the time period for each excitation sourceis the same. In some instances, the time period for each excitationsource is at least or about 50 microsecond (μs), 75 μs, 100 μs, 200 μs,300 μs, 400 μs, 500 μs, 600 μs, 700 μs, 800 μs, 900 μs, 1.0 microsecond(ms), 1.5 ms, 2.0 ms. 2.5 ms, 3.0 ms, 3.5 ms, 4.0 ms, 4.5 ms, 5.0 ms,5.5 ms, 6.0 ms, 6.5 ms, 7.0 ms, 7.5 ms, 8.0 ms, 8.5 ms, 9.0 ms, 9.5 ms,10 ms, 10.5 ms, 11 ms, 12 ms, 13 ms, 14 ms, 15 ms, 16 ms, 17 ms, 18 ms,19 ms, 20 ms, or more than 20 ms. In some instances, the time period isin a range of about 1 ms to about 10 ms. In some instances, thedetection sampling is performed at least or about 1 kHz, 2 kHz, 5 kHz,10 kHz, 15 kHz, 20 kHz, 25 kHz, 30 kHz, 35 kHz, 40 kHz, 45 kHz, 50 kHz,55 kHz, 60 kHz, 65 kHz, 70 kHz, 75 kHz, 80 kHz, 85 kHz, 90 kHz, 100 kHz,120 kHz, 140 kHz, 160 kHz, 180 kHz, 200 kHz, 220 kHz, 240 kHz, 280 kHz,300 kHz, 320 kHz, 340 kHz, 360 kHz, 380 kHz, 400 kHz, 450 kHz, 500 kHz,600 kHz, 700 kHz, 800 kHz, 900 kHz, 1000 kHz, 2000 kHz, 3000 kHz, 4000kHz, or more than 4000 kHz. In some instances, the detection sampling isperformed in a range of about 1 kHz to about 2 MHz. In some instances,the detection sampling is performed in a range of 10 kHz to about 500kHz. In some instances, the detection sampling is performed in a rangeof 20 kHz to about 250 kHz.

In some instances, a first excitation source excites a molecule whoseemission intensity is unrelated to the concentration of any targetmolecule or species to be measured. The excitation source is turned onin between each of the assay intensity measurement sources. In someinstances, the channel is turned on interspersed between the otherchannels when its intensity is above a certain threshold. The channelmay be turned and remains on when its intensity is below the certainthreshold. In some instances, the system comprises time periods where noexcitation source is turned on that are interspersed with time periodswhere a single excitation source is turned on. In some instances, asingle detector is used that is not wavelength dependent. In someinstances, multiple channels are used.

FIG. 94 illustrates a system for spatial multiplexing. In someinstances, multiple excitation sources and/or a controller is used. Insome instances, the excitation filters and/or emission filters areconverted to multiple bandpass filters. In some instances, the multiplebandpass filters substantially pass only those wavelengths correspondingto each of the excitation or emission wavelengths in the system. Thecontroller may control whether the excitation sources are on or off. Insome instances, a wavelength-dispersion element and/or array ofdetectors are added behind the detection restriction. The system 1951comprises a set of excitation sources 1971, 1973, an excitation filter1975, an optical stage 1977, an emission filter 1979 an opticalrestriction (e.g. a pinhole or a slit), a wavelength-dependent disperser1983 and an array of non-wavelength dependent detectors 1985, 1987arranged spatially on an image plane of the wavelength-dependentdisperser 1983. The disperser 1983 changes the angle at which rays ofincoming light substantially impinging on the disperser 1983 at the sameangle leave the disperser 1983. In some instances, an exit angle isdependent on the wavelength of the light impinging on the disperser. Alight source comprising multiple wavelengths may be angularly resolved.In some instances, an angular distribution is created for eachwavelength with a central angle and a measure of dispersion around thecentral angle. At any plane located a finite distance from the exitsurface of the disperser 1983, an image is created where the angulardistribution of light at the exit surface of the disperser 1983 isrealized as a spatial distribution. By choosing a distance for thisplane that is larger than some minimum value, an original sourceconsisting light comprising a finite set of primary wavelengths may beresolved into a spatial distribution where the intensity within anyspatial extent smaller than some fixed figure results primarily from atmost one of the finite sets of primary wavelengths. By arranging thearray of detectors such that the collection surface for each detectorprimarily intersects only one of the spatial extents, awavelength-dependent detector may be created from non-wavelengthdependent detectors. The detector may comprise photodiodes,photomultiplier tubes, silicon photomultipliers, any other opticaltransducer, or any combination thereof. In some instances, the detectorcomprises a photomultiplier (PMT). In some instances, the detectorcomprises a silicon photomultiplier (SiPM) or an array of siliconphotomultipliers. In some instances, the detector is a charge-coupleddevice (CCD) or array of CCDs. The dispersion element may be a grating,a prism, or any object angularly disperses light. In some instances, thedispersion element is a grating. Gratings can be transmissive orreflective. In some instances, reflective gratings comprise a detectorthat is arranged at an angle for intersecting dispersed light.

Disclosed herein are methods for multiplexing using systems as describedherein. For example, a method for multiplexing using the system of FIG.94 comprises excitation sources that are all activated simultaneously.The detector may be sampled at least or about 100 Hz, 500 Hz, 1 kHz, 2kHz, 5 kHz, 10 kHz, 15 kHz, 20 kHz, 25 kHz, 30 kHz, 35 kHz, 40 kHz, 45kHz, 50 kHz, 55 kHz, 60 kHz, 65 kHz, 70 kHz, 75 kHz, 80 kHz, 85 kHz, 90kHz, 100 kHz, 120 kHz, 140 kHz, 160 kHz, 180 kHz, 200 kHz, 250 kHz, 500kHz, 1 MHz or more than 1 MHz.

Systems and methods as described herein for serial flow emulsioncomprises multiplexing of sample, wherein various methods for analyzingmultiplexed samples are used. In some instances, methods for analyzingmultiplexed samples relates to detecting specific droplets (partitions).For example, each wavelength channel has a series of data of detectorintensity as a function of time. For temporal multiplexing, the data maybe obtained by pairing each detector measurement with the excitationsource that was active when the measurement was taken. A subset ofintensity data for each excitation source as a function of time may thenbe generated. For spatial multiplexing, the use of multiple detectorsmay produce a separate subset of intensity data as a function of timefor each channel. As a droplet (partition) enters the optical stage, theintensity signal may increase until the droplet (partition) isapproximately at a center of a region of the optical stage imaged by theoptical restriction, after which the signal diminishes. A singlemeasurement may be determined that represents a peak intensity for eachdroplet (partition). In some instances, a peak-finding algorithm is usedfor detecting the peak. In such an algorithm, the increase in intensityover adjacent signals may be used to determine whether the signal at agiven time is a peak. In some instances, minimum relative or absolutethresholds are applied to the peaks located by a peak-finding algorithm.In some instances, smoothing algorithms are used. Smoothing algorithmsmay be used to reduce or eliminate local fluctuations in the data. Insome instances, the smoothing algorithms are used before applying a peakalgorithm. In some instances, the smoothing algorithms are used afterapplying a peak algorithm.

In some instances, analyzing multiplexed samples comprises methods forcalibration. In some instances, systems and methods for analyzingmultiplexed samples comprise measuring a peak at each channel. In someinstances, if a peak is not detected, the peak is aligned with measuredpeaks. In some instances, calibration comprises examining at time atwhich a droplet (partition) peak was registered on one channel andensuring that each other channel also has a peak at that time. In someinstances, each channel comprises a peak within a fixed time window. Insome instances, the fixed time window is determined when the peak isdetected by another channel.

There are a number of ways of introducing partitions into theinterrogation region. The partitions, coming from the portion of thesystem that prepares them for detection (e.g. in a PCR system, thethermal cycler), will be flowing in a conduit. That conduit may be anysuitable conduit, e.g., a microfluidic channel in a chip, a tube ofround, elliptical, rectangular, or other cross section, or any otherconduit that can contain the partitions. The conduit should be orientedin such a way that the partitions can be illuminated by the excitationenergy and electromagnetic radiation emitted by the fluorophores can bedetected in a photodetector. The conduit may be oriented so that theexcitation energy and the electromagnetic radiation emitted share acommon pathway (“on-axis”), or it can be oriented so that they do notshare a common pathway. For highest signal-to-noise ratio, it ispreferred that the excitation electromagnetic radiation and emittedelectromagnetic radiation do not share a common pathway. For this tooccur, the material comprising the conduit through which the partitionsare passing may not be strongly biased in terms of an illumination oremission direction. For example, when using a channel in a microfluidicchip comprising glass or polymer, illumination energy that is notsubstantially normal to the chip surface will have a relatively highfraction of that energy reflected away from the partitions in the chipwithout exciting the fluorophores. Likewise, emitted energy from thefluorophores in directions substantially deviating in angle from anormal vector to the chip surface will have a substantial fractionreflected or refracted away from those directions as well as absorbed bythe material comprising the chip. To maximize illumination and detectionefficiency, channels contained in microfluidic chips will preferentiallyhave illumination and detection pathways that are on-axis or have asmall angle of deviation between their axes.

In contrast, a conduit comprising a tube (e.g. a tube whose surface hashigher affinity for continuous phase than dispersed phase, such asfluoropolymer tube when the continuous phase is, e.g., a fluorinatedoil) has approximately isotropic illumination and emission frequency inthe azimuthal angle, and so is relatively agnostic as to the directionfor illumination and emission. In this case, off-axis solutions can beused, which, e.g., reduce the amount of stray electromagnetic radiationintroduced into the detection system from the illumination system.Excitation sources can be arranged so that the focal point of theexcitation is on a central point where a partition to be measured ispassing. In one embodiment, the excitation sources are coplanar ornearly so with the photodetector and with each other, with the commonplane substantially orthogonal to the tube through which the partitionsare flowing. In other embodiments, at least one of the excitationsources is offset from the plane orthogonal to the partition conduit;the purpose of doing so would be to increase the number of excitationsources, which may be limited due to geometrical constraints. Wherepossible, the preferred embodiment is one where all of the sources arecoplanar or nearly so with a plane orthogonal to the partition tube,because that will minimize reflection/refraction of excitation energyincident on the tube. Thus in certain embodiments the systems andmethods provided herein include a detector comprising a plurality ofexcitation sources situated within 40, 30, 20, 15, 10, 5, 2, or 1 degreeof a plane that is orthogonal to a conduit, for example, a tube,illuminated by the excitation sources. In certain embodiments, one ormore photodetectors is also situated within 40, 30, 20, 15, 10, 5, 2, or1 degree of the plane that is orthogonal to the conduit.

The cross-section of the detection conduit may be larger, the same as,or smaller than the equivalent average spherical cross-section of thepartitions. Thus, the cross-sectional area of the conduit may be anysuitable cross-section, depending on the equivalent average sphericalcross-section of partitions. For example, for partitions that have anaverage diameter of 100 um, the average spherical cross-sectional areawill be ˜7850 um², and appropriate calculation may be made of thecross-sectional area of the conduit, depending on whether the conduit isto be larger, smaller, or the same cross-sectional area as the averageequivalent spherical cross-section of the partitions. Exemplarycross-sectional areas for the conduit in the interrogation region are10-1,000,000 um², 10-100,000 um², 100-100,000 um², 100-50,000 um²,100-40,000 um², 500-50,000 um², 500-40,000 um², 1000-100,000 um²,1000-50,000 um², 1000-20,000 um², 2000-20,000 um², or 2000-10,000 um².

In certain embodiments, the cross-section of the interrogation region issmaller than or the same as the equivalent average sphericalcross-section of the partitions. This ensures that partition positionwithin the cross-section of the conduit does not affect the intensitymeasurement, because the partition consumes the entire cross section, orsubstantially the entire cross-section, of the conduit; it will beappreciated that in embodiments in which the surface of the conduit hasgreater affinity for continuous phase than for dispersed phase, a thinlayer of continuous phase will be associated with the walls of theconduit and thus a partition flowing through the conduit will have across-section that is slightly smaller than that of the conduit. Incertain embodiments, the cross-section of the conduit is smaller thanthat of the equivalent average spherical cross-section of thepartitions. When this is the case, the partition becomes elongated inthe conduit and, when the sampling frequency is at least twice as highas the frequency of partition arrival at the center point of thedetection region in the conduit, multiple measurements may be made ofeach partition. By using the number of measurements above some minimumintensity in conjunction with the cross-section of the conduit and theliquid volumetric flowrate in the conduit, the size of the partitionsmay be calculated. This information may, in turn, be used to calculatevarious quantities about the components measured within the partitions(e.g. partition volumes, component concentrations, Poisson correctionsbased on the underlying distribution of partition sizes, etc.) and/ormay be used to accept or reject partitions for quality reasons (e.g. toobig or too small). Thus in certain embodiments, the detection conduithas a cross-sectional area that is not more than 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or 95% and/or at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the equivalent average spherical cross-section ofpartitions of dispersed phase flowing through it. The equivalent averagespherical cross-section of the partitions is, e.g., the averagecross-section of the partitions if their volume of each is contained ina sphere.

To perform assays with the system, it is important to measure and assignan emissive intensity in each wavelength range of interest to eachpartition. One such way to do this is to collect a relatively largefraction of the electromagnetic radiation emitted from the partitionsilluminated in the interrogation region and to make mathematicalcorrections to the intensity based on the previous and subsequentmeasurements made on the partitions ahead of and behind the partition inthe conduit, respectively. Another way to do this is to restrict theelectromagnetic radiation reaching the photodetector(s) so thatsubstantially only that electromagnetic radiation emitted from a singlepartition at a given time reaches the photodetector(s). This trades theefficiency of electromagnetic radiation collection (i.e. the fraction ofthe emitted electromagnetic radiation measured at a photodetector) forreduction in signal from other partitions than the partition beingmeasured and has the potential to make data analysis simpler; it alsoenables the partition measurement technique described above. Methods toachieve this include placing an optical restriction (e.g. a slit orpinhole) between the conduit containing the partitions and thephotodetection element(s). The size of the optical restriction willdepend on its location in the optical path, but the intention is toreduce the field of view, e.g., so that only or substantially onlyelectromagnetic radiation from a single partition is measured by thephotodetector(s). The desired size and location of the opticalrestriction will depend on, e.g., the degree of separation of partitionsof dispersed phase by continuous phase as they pass through thedetection region (the greater the separation, the more electromagneticradiation the optical restriction can let through from a given partitionwithout allowing electromagnetic radiation from adjacent partitionsthrough), the narrowness of the conduit in the detection region(conduits narrow enough to cause elongation of the partitions alsoessentially separate the center of one partition from others, decreasingthe amount of electromagnetic radiation from others).

Thus, provided herein are systems and methods for detection of a single(partition) at a time, wherein the detection may comprise use of anoptical stage. In some instances, a single droplet (partition) ismeasured by restriction of the excitation dimension and emissiondimension of the optical stage. In some instances, the restriction is ageometric restriction. For example, geometric restrictions include useof a pinhole, slit, or any arrangement of openings in a surface thatrestrict the geometric and/or angular extent of light entering andexiting the optical stage and/or impinging on the photodetectionelement. In some instances, the restrictions allow for only the droplet(partition) to be detected. Following collection of emitted light, animage of the optical stage may be projected on a plane in front of thedetector. By using a restriction in that plane such as a pinhole or slitor combination thereof, only a portion of the image comprising thedroplet (partition) may be captured. In some instances, the restrictionis located in a plane between the detector and its lens. In someinstances, the restriction is located in front of the optical stage. Insome instances, multiple restrictions are used. In some instances, atleast or about 1, 2, 3, 4, 5, 6, or more than 6 restrictions are used.In some instances, the emitted light is at least or about 0.5 mm², 0.75mm², 1.0 mm², 1.25 mm², 1.5 mm², 1.75 mm², 2.0 mm², 2.5 mm², 3.0 mm²,3.5 mm², 4.0 mm², or more than 4.0 mm².

Hence, the detector may further comprise a pinhole (e.g. opticalrestriction). In some instances, the pinhole reduces the spatial extentof the excitation light source. In some instances, the pinhole allowsthe excitation light source to only illuminate one droplet (partition)in the optical stage at a given point in time. In some instances, thepinhole allows one droplet (partition) to be measured.

An exemplary diagram of a detector comprising an optical restriction isillustrated in FIG. 90. The detector comprises a light source (the“excitation” source) 901 to illuminate droplets (partitions) movingthrough an optical stage 903 and a photo-detector 907 to collect emittedlight from the droplets (partitions) which corresponds to a fluorescentmarker. FIG. 91 shows a detailed arrangement of FIG. 90. The detectorcomprises an excitation filter 1003 on the light source (the“excitation” source) 1001 and an emission filter between the opticalstage 1007 and the photo-detector 1011. The filter for the excitationsource may allow at least one wavelength range to pass through it. Insome instances, the at least one wavelength that passes through thefilter of the excitation source corresponds to an excitation wavelengthof at least one fluorescent marker in a reaction or assay. In someinstances, the filter for the photo-detector allows at least onewavelength range to pass through it. In some instances, the at least onewavelength that passes through the filter of the photo-detectorcorresponds to an emission wavelength of at least one fluorescent markerin a reaction or assay. In some instances, the filters improve asignal-to-noise ratio. In some instances, the signal-to-noise ratio isimproved by only passing wavelengths that correspond to the reaction orassay.

An further exemplary diagram of a detector comprising an opticalrestriction, e.g., pinhole is illustrated in FIG. 92. Excitation lens1105 on the excitation filter 1101 and emission lens 1109 on theemission filter 1111 allow for light to focus on the pinhole on theexcitation side and collimate emission light such that light passesthrough the filter substantially normal to the filter surface.

The signal measured by the photodetector(s) (once demodulated, in thecase of the lock-in detection system) is a time series of valuescorrelated to the intensity or power of electromagnetic radiationincident on the photodetector. These values rise and fall as partitionspass through the system, with each partition having a relative maximumintensity value as the partition passes the portion of the conduit withhighest optical collection efficiency. For many assays, it is importantto assign a single value for each wavelength (or range of wavelengths)to each partition. As such, the time series data must be transformedinto signal data in each wavelength (or wavelength range) as a functionof partition number. This can be achieved with suitable peak findingalgorithms. Peak finding can be complicated by noise in the data, whichcan be expressed as multiple relative maxima and minima within aspecific partition. Smoothing the data can reduce the complexity of peakfinding. Any suitable data smoothing technique may be used. Exemplarydata smoothing techniques include convolution (1-D, 2-D, 3-D) with anaveraging kernel (e.g. a Gaussian, a hat function, an impulse, a unitstep function), moving averages, regression-based curve fitting to amodel function, frequency-space transforms or filters with removal ofhigh-frequency components (e.g. Butterfield filter, etc.).

Thus, systems and methods as described herein for detecting droplets(partitions) may comprise various algorithms. In some instances, thealgorithms are peak-finding algorithms. The algorithms may be applied toa single wavelength channel or an accumulation of multiple wavelengthchannels. In some instances, a number of droplets (partitions) areknown. In some instances, the number of droplets (partitions) is knownbecause the average droplet size and total injection volume size isknown. By counting the droplets (partitions), a sample may be delineatedby the total droplet (partition) number reaching some minimum number ofdroplets (partitions). In some instances, samples with deviateddistributions are identified.

In some instances, groups of droplets (partitions) are detected bymeasuring the number of distinct peaks with signal intensities above abaseline signal intensities, assigning the peaks to individual droplets(partitions), and counting the total number of droplets (partitions)observed in a time period. In some instances, by knowing a volumetricflow rate and injection volume, the group of droplets (partitions)comprises a minimum time period following detection of a first signalabove the minimum threshold. A second group of droplets (partitions) maybe determined by seeing a second signal above a minimum threshold.

In a system that includes an injector, discrete packets of samples canbe injected into the system and individually partitioned intopartitions. It can be important to be able to discriminate partitionsthat belong to a first sample from partitions that belong to a secondsample. This can be done by, e.g., using intensity measurements from thephotodetectors. For example, if a spacing fluid (e.g. a silicone-basedor mineral oil) is added between injections of samples to be quantified,measurements made on the spacing fluid can provide information aboutwhere a first sample ends and a second sample begins. In certainembodiments, the spacing fluid does not contain a fluorophore capable ofbeing excited by the illumination sources in the detector, thephotodetectors should measure a relatively low intensity while thatspacing fluid is moving through the detector. A period of continuous lowintensity indicates a boundary between the first sample and the secondsample. In certain embodiments, at least one dye excitable by theillumination sources in the detector is added to the spacing fluid. Ifthe emission frequency for this dye overlaps with the emission frequencyfor dyes related to probes for analytes in the system, an extendedperiod of high intensity for that dye (or dyes) indicates a boundarybetween the first sample and the second sample. If the spacing fluid isincapable of forming stabilized partitions near the size of the aqueouspartitions in the continuous phase, this extended period should be muchlonger than the period of increased intensity representing a partition.Alternately, if the fluorophore in the spacing fluid has an emissionband that does not substantially overlap (in wavelength) with theemission range of the other dyes in the aqueous phase, detection of asignal in this wavelength band will be enough to determine that a sampleboundary has been reached. By choosing combinations of fluorophores (orthe absence of any excitable fluorophores), samples can be labeled bythe spacing fluid. For example, if two spacing fluids are available forinjection, one without a fluorophore and one with a fluorophore, thereare three combinations of labels that can be used to discriminate thesamples arriving at the detector from each other. By alternating thesespacing fluids, a problem in injection can be detected if two equivalentlabels are detected in immediate series. If three spacing fluids areavailable for injection, one each with a different fluorophore (orpossibly one without), seven labels are available if more than onespacing fluid may be injected between each sample. Alternatively oradditionally, there can be just use one spacing fluid between eachpacket. Since there will be a spacing fluid before and after eachpacket, the data from both can be used to barcode the sample. Forexample, if 4 dyes are used with a 4-color detector, it is possible tobarcode 256 samples with binary intensity and 6561 samples with trinaryintensity. If 3 dyes are used with a 3-color detector it is possible tobarcode 64 samples with binary intensity and 726 samples with trinaryintensity. Alternatively or additionally, the volume of dyes (translatedto a length of a high signal at the detector) can provide additionaldiscrete states, expanding the number of unique states. Using the datafrom both increases sample injection speed since only one spacing fluidis needed. Suitable dyes and spacing fluid could be contained, e.g., ina consumable plate, e.g., in a second plate holder and the system can bedesigned, e.g., to include or not include it.

Thus, systems and methods as described herein may be used to detectgroups of droplets (partitions). In some instances, the groups ofdroplets (partitions) represent different injections of dispersed phases(e.g. sample assays). In some instances, a first group of droplets(partitions) is distinguished from a second group of droplets(partitions) by the sequence of injection. For example, a purge fluid(spacer fluid) can be injected into the microfluidic path or channelbetween dispersed phases. In some instances, the purge fluid (spacerfluid) does not comprise fluorescent markers of the assay or reaction.The first group of droplets (partitions) may be distinguished from thesecond group of droplets (partitions) due to the purge fluid (spacerfluid) that is injected between the first group of droplets (partitions)and the second group of droplets (partitions) not emitting an opticalsignal above a threshold level within a specified wavelength range.

In some instances, groups of droplets (partitions) are detected using afluorescent marker that is excited and/or emits light at a differentwavelength than fluorescent markers in a dispersed phase. Thefluorescent market may be miscible with the continuous phase butimmiscible with the dispersed phase. In some instances, detection of thefluorescent wavelength at the different wavelength than that emitted byfluorescent markers in a dispersed phase indicates that no droplets(partitions) of the dispersed phase are passing through the detector. Insome instances, a fluorescent marker is added to the purge fluid and/orseparation (spacer) fluid between the dispersed phases. In someinstances, the fluorescent marker is miscible with the purge fluidand/or separation fluid and immiscible with the dispersed phase. In someinstances, the fluorescent marker is used to detect concentration ofspecies in the dispersed phase.

In some instances, groups of droplets (partitions) are detected using afluorescent marker that is emitted at a similar wavelength than adispersed phase. In some instances, the fluorescent marker with anemission wavelength similar to the wavelength of the disperse phase isused to determine concentration of species in the dispersed phase. Forexample, the florescent marker is added to a purge fluid and/orseparation fluid at a concentration resulting in an optical signalhigher than the optical signal of droplets (partitions) of the dispersedphase. In some instances, the optical signal of the fluorescent markeris at least or about 0.5×, 1.0×, 1.5×, 2.0×, 2.5×, 3.0×, 3.5×, 4.0×,4.5×, 5.0×, 5.5×, 6.0×, 6.5×, 7.0×, 7.5×, 8.0×, 8.5×, 9.0×, 9.5×, or 10×higher than the optical signal of the droplets (partitions) of thedispersed phase. In some instances, droplets (partitions) aredistinguished from the purge fluid and/or separation fluid based on thedifference in optical signal. In some instances, a number of opticalsignals of the purge fluid and/or separation fluid are detected betweendispersed phases.

Partitions arriving at the detector may be measured as they enter theinterrogation region. Due to fluidic restrictions, these partitions maybe very close together. In this situation, signals will not follow amovement from baseline up to peak value for partitions, because foralmost the entire time measurements are taken, partitions will be at thefocal point in the system. Instead, there will only be a small breakbetween partitions where the signal will be reduced. In this situation,the magnitude of the signal reduction will indicate whether thepartition was a “positive” or a “negative” partition, and peak-findingmethods may be used that either find “valleys” in the signal or operateon the inverse of the signal.

Alternatively, it may be desirable to separate partitions out by addingcontinuous phase to the system and/or by narrowing the conduit throughwhich partitions flow. This can be performed ahead of the detector in anumber of ways. In certain embodiments, another conduit may join themain flow conduit and add fluid, e.g., continuous phase, around thepartitions, separating them. In certain embodiments, these conduits arepart of a microfluidic device. The conduits can be machined out of afirst substrate using an appropriate method (e.g. milling or othersuitable method) and joined to a second substrate that contains at leastone inlet port and at least one outlet port. The first and secondsubstrate may be composed of one or more of a variety of materials,including but not limited to glass, silicon, or one or more polymers. Incertain embodiments, the polymers comprise a fluoropolymer and thecontinuous phase comprises a fluorinated oil. In another embodiment, thefirst or second substrate (or both) are composed of glass coated with acoating comprising a fluoropolymer. One method of joining the first andthe second substrate so that leaks do not occur is to form a chemicalbond. In the case that the first and second substrate arefluoropolymers, a bonding method may comprise a step that removes atleast some of the fluorine from the surface of the fluoropolymers, thusallowing a chemical bond to be formed. Another method of joining thefirst and second substrate together includes machining a surface of thefirst or second substrate such that only a small relief of the first orsecond substrate is available to contact the opposing substrate. Aleak-resistant seal can be created by applying mechanical force toconnect the first and second substrate together, that mechanical forcecreating pressure at the interface between the first and the secondsubstrate. Another method of producing a leak-resistant seal is to usean elastomeric sealing material (e.g. an o-ring) to create a sealbetween the first and the second substrate. While this seal may notallow perfect contact between the first and the second substrate, if theeffective space between the substrates is small enough and thesubstrates have a lower affinity for the dispersed phase (i.e. thepartitions) than the continuous phase, substantially no dispersed phasewill be able to flow through the space between the substrates and“short-circuit” the conduits.

In certain embodiments, conduits in the microfluidic device can bemachined into a single substrate. One example of this is to drillcoplanar conduits of cylindrical or substantially cylindricalcross-section into the substrate. These conduits can meet in a t- orcross-arrangement, or in any other suitable geometric relationship. Incertain embodiments, the exit conduit may not be co-planar to the atleast one inlet conduit.

In certain embodiments, continuous phase added to the system, e.g.,partition spacing oil, is collected by removing, e.g., siphoning, excessoil after partition formation and reintroducing that partition oil forspacing purposes just before detection. See description elsewhere hereinAny suitable method may be used for removing and/or reintroducingcontinuous phase. In certain embodiments, a small pored substrate isplaced inline after the partition splitting junction. The pores aresubstantially small enough that a partition will not enter the oilsiphon line and continue down the conduit, e.g., to a reactor such as athermocycler. That continuous phase, e.g., oil, siphon line is thenreintroduced in the separation system to provide a separation betweeneach partition for detection. This evacuation and reintroduction of theseparation continuous phase, e.g., oil, between partitions reduces thepartition velocity during reaction, e.g., thermocycling, increasing thepartition stability and reducing the tubing length required in thereactor, e.g., thermocycler.

Additionally or alternatively, continuous phase, e.g., partition oil,may be introduced before detection and then removed, e.g., siphoned,just after detection. This removed, e.g., siphoned continuous phase,e.g., oil may be transported to waste and/or recycled into a spacingcontinuous phase, e.g., oil, vessel for continuous partition separation.

Thus, in some instances, a microfluidic chip may be used to separate thedroplets. The droplets (partitions) may then leave the microfluidic chipand enter a microfluidic tube that serves as the optical stage.Connections between the microfluidic chip and the microfluidic tube orchannel or connections between the microfluidic chip and the opticalstage tube may be made so that there are few or no dead flow regions fordroplets. In some instances, droplets (partitions) are separated on achip with either a t-junction or a v-junction (see below for v-junctionembodiments). In some instances, a constricted region is continuous froma tubing interface to a chip. In some instances, the tube forms theoptical stage. Thus, in some instances, the droplets (partitions) areseparated from each other axially as they pass through the opticalstage. In some instances, the droplets (partitions) are separated byaddition of a continuous phase between each droplet (partition). In someinstances, the continuous phase comprises a surfactant. In someinstances, the continuous phase does not comprise a surfactant.Exemplary surfactants include, but are not limited to, a fluorocarbon, ahydrocarbon, a silicone, or an oil.

Additionally or alternatively, partition separation is achieved byreduction in the conduit dimensions; this alone can achieve greaterseparation of partitions, or it can be used in conjunction with a systemthat adds separation fluid, e.g., continuous phase. Since a conduitdimension reduction results in a volumetric reduction and there is aminimum amount of interstitial continuous phase, e.g., oil, between eachpartition, the volume reduction results in an increase in thepartition-to-partition distance in the confined conduits. In certainembodiments, these conduits are constructed in an optically transparentsubstrate. In certain embodiments, partitions enter the constrictedconduit, enter a partition imaging chamber sized to fit exactly orsubstantially exactly one partition, and then exit the chamber in asecond constricted conduit. This allows for a single partition to beinterrogated by the optical system while maximizing the spacing betweeneach consecutive partition before and after the imaging chamber. See,e.g., FIG. 65. This partition imaging chamber can be fabricated in anysuitable material, for example, fabricated in glass substrate coatedwith a fluorophilic surface chemistry.

Partitions arriving at the detector may be measured as they enter theinterrogation region. Due to fluidic restrictions, these partitions maybe very close together. In this situation, signals will not follow amovement from baseline up to peak value for partitions, because foralmost the entire time measurements are taken, partitions will be at thefocal point in the system. Instead, there will only be a small breakbetween partitions where the signal will be reduced. In this situation,the magnitude of the signal reduction will indicate whether thepartition was a “positive” or a “negative” partition, and peak-findingmethods may be used that either find “valleys” in the signal or operateon the inverse of the signal.

Thus, systems and methods provided herein can achieve a separation ofpartitions of dispersed phase flowing in continuous phase prior todetection, where prior to separation, the average distance betweenpartitions of dispersed phase in the continuous phase is a, and afterseparation, the average distance between partitions is b, where b>a.See, e.g., FIG. 51 for one way that a and b can be measured, e.g., asdistance between geometric centers of partitions, such as distancebetween centers of spheres. It will be appreciated that in certain casespartitions alter geometry in going from an area before separation to anarea after separation; for example, in certain embodiments separation isachieved by narrowing the conduit through which partitions flow to across-sectional area equal to or less than the average cross-sectionalarea of the partitions, and partitions may alter geometry from sphericalto elongated. In such a case, a or b may be determined for what thedistance would be if both partitions were spherical, so that, e.g.,elongation of partitions does not in itself increase the measure butrather actual spacing between partitions increases the measure. Incertain embodiments, b is at least 102, 105, 110, 125, 150, 175, 200,225, or 300% of a, and/or b is at most 105, 110, 125, 150, 175, 200,225, 300 or 400% of a.

FIG. 52 (t-junction chip-based separator). An embodiment of a system forseparating flowing dispersed phase partitions is shown in FIG. 52. Thesystem comprises a substrate 5201 that comprises a main channel 5202, aninlet to the main channel 5203, and outlet to the main channel 5204, abranch channel 5206, and an inlet to the branch channel 5207 where thebranch channel intersects the main channel within the substrate. A firstcontinuous phase and partitions of dispersed phase enter through inlet5203, while a second continuous phase is added at inlet 5207. In certainembodiments, the first continuous phase and second continuous phase havethe same composition. In certain embodiments, the first continuous phasecomprises an oil and a surfactant, while the second continuous phasedoes not comprise more than 5, 4, 3, 2, 1.5, 1.2, 1.1, 1.0, 0.9, 0.8,0.7, 0.5, or 0.2% surfactant, e.g., not more than 2% surfactant, such asnot more than 1% surfactant; this may be done to, e.g., save the cost ofthe surfactant. Continuous phases, oils, and surfactants are describedin more detail elsewhere herein. The substrate 5201 may be any suitablematerial in which small channels may be manufactured. The surfaces ofthe substrate 5201, in particular, surfaces which contact flow ofdispersed phase, can comprise a material that has a higher affinity forthe continuous phase than for the dispersed phase. In certainembodiments, the first and second continuous phase comprise fluorinatedoils and the surface of the substrate 5201, in particular, surfaceswhich contact flow of dispersed phase, comprises a fluoropolymer. Anysuitable method may be used to fabricate the system; in some cases,methods similar or identical to those discussed for partitioners may beused. In certain embodiments, at least one of the main or branchchannels is formed by creating relief features in at least one planarsurface of the substrate and joining that surface to a second planarsurface of the substrate to create an enclosed channel or set ofchannels. Suitable means of creating the relief include, but are notlimited to, endmilling, laser machining, etching, photolithography. Incertain embodiments, at least one of the main or branch channels areformed by creating hole features in a monolith of the substrate. Thehole features can be created by any suitable method, such as drilling,e.g., mechanical drilling, laser drilling, etc.

The flow of first continuous phase and dispersed phase partitions issupplemented by flow of second continuous phase at the junction of themain and branch channels 5205. The cross-sectional area of at least oneof the main or branch channels may be larger, smaller, or equal to theequivalent spherical cross-sectional area of the partitions flowingthrough the channel. For example, in channels with circularcross-sections, the diameter of at least one of the main or branchchannels may be larger, smaller, or equal to the equivalent sphericalcross-sectional diameter of the partitions flowing through the channel.In certain embodiments, the cross-sectional area of the main channel atthe junction 5205 is larger than the equivalent sphericalcross-sectional area of the dispersed phase partitions flowing throughthe main channel (e.g., diameter of main channel is greater than averagespherical diameter of partitions, if the cross-section of the channel iscircular or nearly circular), allowing the second continuous phase to beadded to the space between more than one partition at a time.

The combined flow of dispersed phase partitions, first continuous phase,and second continuous phase exits the substrate at the main channeloutlet 5204, where it enters a tube 5208. The tube passes through aninterrogation region 5209, where at least one property of the dispersedphase partitions is detected. The tube is at least partially transparentto electromagnetic radiation in a first wavelength range and a secondwavelength range, such that electromagnetic radiation incident on thetube in the first wavelength range may pass through the tube and, e.g.,excite a reporter molecule in dispersed phase partitions positionedwithin the interrogation region 5209, with electromagnetic radiationemitted by the molecule in the second wavelength range may pass out ofthe tube to be detected. In some embodiments, the tube material allowsat least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% and/or not morethan 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of theelectromagnetic radiation in the first wavelength range to pass throughit and least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% and/or not morethan 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of theelectromagnetic radiation in the second wavelength range to pass throughit. The tube may be constructed of any suitable material that meets thedescribed requirements for translucence. In certain embodiments, thesurface of the tube comprises a material that has a higher affinity forthe first continuous phase, the second continuous phase, or both than ithas for the dispersed phase. In certain embodiments, the cross-sectionalarea of the interrogation region of the tube is equal to or smaller thanthe cross-sectional area of the dispersed phase partitions, such thatthe dispersed phase partitions are in single file and they occlude allor substantially all of the cross-sectional area of the channel; inembodiments in which channels have greater affinity for continuous phasethan dispersed phase, a thin layer of continuous phase is in contactwith the wall, so that partitions occlude all of the channel except forthe continuous phase film layer. This reduces optical contributions frommore than a single dispersed phase partition.

Systems and methods as described herein may allow for estimation ofdroplet (partition) size. In some instances, systems and methods forestimating droplet (partition) size comprise detecting light emitted bya single droplet (partition). In some instances, systems and methods forestimating droplet (partition) size relate to a cross-section of achannel of an optical stage in which the channel of the optical stage issmaller than a cross-section of the droplet (partition). In someinstances, as a result of cross-section of a channel of an optical stagebeing smaller than a cross-section of the droplet (partition) thedroplet (partition) substantially fills the channel in the opticalstage.

A length of a droplet (partition) may be estimated as it passes throughthe optical channel. For example, when a droplet (partition) enters theoptical stage and is detected by the detector, a signal of the detectorwill rise above a minimum threshold and will remain above a minimumthreshold until the droplet (partition) passes out of the optical stageand is no longer detected by the detector. Once the droplet (partition)passes out of the optical stage is no longer detected by the detector,the signal may drop. An amount of time from the signal being above aminimum threshold and the signal dropping may be correlated to thelength of the droplet (partition). In some instances, a correction isapplied. In some instances, the correction is due to velocity of flow inthe channel. In some instances, the correction is applied based on alength or transit time of the droplet (partition) as the droplet(partition) pass through the optical stage. In some instances, the timeis used to determine a droplet (partition) size. For example, the timethat the droplet (partition) passes through the optical stage ismultiplied by a volumetric flow rate to determine droplet (partition)size. Other correction values may be applied in determining droplet(partition) size. In some instances, a laminar flow correction isapplied.

Exemplary droplet (partition) volume measured using systems and methodsas described herein include, but are not limited to, at least or about 5micron (um), 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90um, 100 um, 120 um, 140 um, 160 um, 180 um, 200 um, or more than 200 um.In some instances, droplet (partition) sizes are at least or about 200um, 300 um, 400 um, 500 um, 600 um, 700 um, 800 um, 900 um, 1000 um, ormore than 1000 um.

Connections between the droplet (partition) flow channel and themicrofluidic channel or tube can result in little or no dead volume. Anexemplary connection is a press-fit or compression connection asdescribed herein. The droplet (partition) flow channel can narrow to theoptical stage diameter at a t-junction with the continuous phasechannel. The narrowed droplet flow channel extends beyond the t-junctionto provide sufficient space for the optical excitation and detectionsystem to be implemented. The distance between droplets (partitions)following the t-junction is greater than the distance between thedroplets prior to entering the t-junction.

FIGS. 53A and 53B—On-chip interrogation region FIGS. 53A and 52B showdifferent views of another embodiment of a dispersed phase partitionseparator and an interrogation region. The embodiment comprises asubstrate 5301, a main channel 5302 comprising an inlet 5303 and anoutlet 5304, a branch channel 5305 comprising an inlet 5306 and aninterrogation region 5307. A first continuous phase and partitions of adispersed phase enter the main channel 5302 through the inlet 5303 and asecond continuous phase enters the branch channel 5306. The main channeland the branch channel meet at a junction 5308, where the addition ofsecond continuous phase increases the average spacing between partitionsof the dispersed phase in the channel. The dispersed phase partitionstravel through the interrogation region 5307, where a property of thedispersed phase partitions is detected. In some embodiments,electromagnetic radiation of a first wavelength of light impinges on themain channel 5302 in the interrogation region 5307 and excites amolecule in the dispersed phase partitions, which emits electromagneticradiation of a second wavelength of electromagnetic radiation. In someembodiments, the material and/or geometry of the interrogation regionallows at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% and/or notmore than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of theincident electromagnetic radiation in the first wavelength range to passthrough it and least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% and/ornot more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of theemitted electromagnetic radiation in the second wavelength range to passthrough it. In certain embodiments, the cross-sectional area of theinterrogation region 5307 of the main channel 5302 is equal to orsmaller than the cross-sectional area of the dispersed phase partitions,such that the dispersed phase partitions are in single file and theyocclude all or substantially all of the cross-sectional area of thechannel; in embodiments in which channels have greater affinity forcontinuous phase than dispersed phase, a thin layer of continuous phaseis in contact with the wall, so that partitions occlude all of thechannel except for the thin continuous phase film layer. This reducesoptical contributions from more than a single dispersed phase partition.Additionally, partitions that have an equivalent sphericalcross-sectional area substantially the same or greater than thecross-sectional area of the main channel 5302 in the interrogationregion 5307 reduce variability in detected optical signals due todispersed phase position within the channel. Partitions with anequivalent spherical cross-sectional area greater than or equal to thecross-sectional area of the main channel in the interrogation region5307 will become distorted so as to be longitudinally distributed alongthe channel. By measuring an optical property of a dispersed phasepartition from when it enters the interrogation region 5307 to when itleaves the interrogation region 5307 and combining it with otherinformation about the dispersed phase partition flow, a size property ofthe dispersed phase partition may be calculated. In certain embodiments,the cross-sectional area of the main channel at the junction 5308 islarger than the equivalent spherical cross-sectional area of thedispersed phase partitions (e.g., diameter of main channel is greaterthan average spherical diameter of partitions, if the cross-section ofthe channel is circular or nearly circular), allowing second continuousphase to be added between more than one pair of dispersed phasepartitions simultaneously. In certain embodiments, the main channel 5307reduces in cross-sectional area (e.g., reduces in diameter if thechannel has a circular or nearly circular cross-section) after thejunction 5308 before entering the interrogation region 5307 so as toreduce the cross-sectional area of the main channel 5302 in theinterrogation region 5307 to a value equal to or below the equivalentspherical cross-sectional area of the dispersed phase partitions. Incertain embodiments, the cross-sectional area of the main channel 5302in the junction 5308 is less than or equal to the equivalentcross-sectional area of the dispersed phase partitions and remains sothrough the interrogation region 5307. The first continuous phase,second continuous phase, and partitions of dispersed phase exit the mainchannel 5302 at the outlet 5304. The cross-sectional area of the mainchannel 5304 beyond the optical stage may increase beyond thecross-sectional area of the main channel 5302 in the interrogationregion 5307, to, e.g., facilitate fluidic connections. Thecross-sectional profile of the main channel 5302 or branch channel 5306may be any suitable cross-sectional profile. In some embodiments, one ormore of the channels have a rectangular, semi-circular, or circularcross-sectional profile.

In certain embodiments, the first continuous phase and the secondcontinuous phase have the same composition. The first continuous phase,the second continuous phase, or both can comprise a fluorinated oil. Incertain embodiments, the first continuous phase additionally comprises asurfactant to stabilize dispersed phase partitions in the firstcontinuous phase. In certain embodiments, the first continuous phasecomprises an oil and a surfactant, while the second continuous phasedoes not comprise more than 5, 4, 3, 2, 1.5, 1.2, 1.1, 1.0, 0.9, 0.8,0.7, 0.5, or 0.2% surfactant, e.g., not more than 2%, such as 1%surfactant; this may be done to, e.g., save the cost of the surfactant.Continuous phases, oils, and surfactants are described in more detailelsewhere herein. In some embodiments, the surface of the substrate5301, in particular, surfaces which contact flow of dispersed phase,comprises a material that has a higher affinity for at least one of thecontinuous phases than for the dispersed phase, reducing the likelihoodthat dispersed phase will wet the surfaces of the substrate 5301. In apreferred embodiment, the entire substrate 5301 comprises a materialthat has a higher affinity for at least one of the continuous phasesthan for the dispersed phase. In a certain embodiments, the surface ofor entire substrate 5301 comprises a fluoropolymer. The channels 5301and 5306 may be formed in the substrate by any suitable method, asdescribed elsewhere herein.

Thus, in some instances, the droplet (partition) separation and anoptical stage are combined. The droplet (partition) separation andoptical stage may be on the same microfluidic chip. The emulsion streamcoming from the rest of the system, e.g. a reactor such as a thermalcycler may enter a first channel on the chip. A stream of continuousphase may enter a second channel on the chip. In some instances, the twochannels both constrict and then meet at a t-junction. The combinedstreams flow through an extended region at the same constricted size, aportion of which may comprise the optical stage. The constricted channelmay then expand to a larger diameter and leaves the chip through, e.g.,a press-fit connection. In some instances, the first channel and thesecond channel do not constrict before meeting in a t-junction. In someinstances, a constriction occurs beyond the exit of the t-junction. Insome instances, the constriction is smaller than the characteristic sizeof the droplets so that a droplet (partition) substantially fills thechannel in the optical stage. A portion of the constricted channel mayform the optical stage. In some instances, the channel then expands to alarger size so that it substantially matches the cross-section of anexit tube. In some instances, the junction is a “v-junction.” In someinstances, the streams are combined at an angle substantially less than90 degrees, for examples, at least or about 10 degrees, 15 degrees, 20degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, 50degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees, or80 degrees

In some instances, a reverse-v junction is used with a tubular opticalstage. In some instances, an exit of the reverse-v junction is tubularand continues to form an optical stage of the detector.

FIG. 54—Collision-style separator FIG. 54 shows a system of channels forincreasing the spacing between partitions of dispersed phase. The systemcomprises a first inlet channel 5401 for a flow comprising a firstcontinuous phase and partitions of at least one dispersed phase, asecond inlet channel 5402 for a flow comprising a second continuousphase, and an outlet channel 5403 where the combined flow of the firstinlet channel 5401 and second inlet channel 5402 exits. The first inletchannel 5401 and second inlet channel 5402 meet at a junction 5404,where their respective flows are combined into the flow of channel 5403.The channels 5401 and 5402 can have axes that are offset by an angle nogreater than 45 degrees; in certain embodiments, the channels 5401 and5402 are co-axial or substantially co-axial At the junction 5404, thesecond continuous phase is added between partitions of the firstdispersed phase such that the spacing between the partitions of thedispersed phase is, on average, increased. The cross-sectional area ofthe first inlet channel 5401 and second inlet channel 5402 can be thesame or substantially the same, an arrangement that may, e.g., simplifymanufacturing. For example, the channels may be created in a substrateby a single set of machining operations. In certain embodiments, thecross-sectional area of the first inlet channel 5401 at a point justahead of the junction region 5404 can be, e.g., equal to or less thanthe equivalent spherical cross-sectional area of the dispersed phasepartitions such that the dispersed phase partitions enter the junctionregion 5404 in single file or substantially in single file. In such anembodiment, the second continuous phase may be added between thepartitions of the first dispersed phase in a more controlled manner,making the spacing between partitions of the first dispersed phase moreuniform. In certain embodiments, the outlet channel 5043 has across-sectional area equal to or less than the equivalentcross-sectional area of the dispersed phase partitions so as to onlyallow a single partition into the outlet channel 5403 at any one time.This may make the spacing between the dispersed phase partitions moreuniform. The cross-sectional area of the outlet channel 5403 mayincrease to a size larger than the equivalent cross-sectional area ofthe dispersed phase partitions at some distance beyond the junction5404. In some embodiments, this distance may be at least 1×, 2×, 3×, 4×,5×, or 10×, and/or not more than 2×, 3×, 4×, 5×, 10×, or 15× theequivalent circular diameter of the cross-sectional area of the outletchannel 5403 at the junction 5404.

The embodiment shown in FIG. 54 may be constructed by any suitablemethod. In certain embodiments, the first inlet channel 5401, secondinlet channel 5402, and outlet channel 5403 may comprise tubing. Incertain embodiments, the first inlet channel 5401 and second inletchannel 5402 may be formed in a substrate and the outlet channel 5403may comprise a tube that is connected to a hole in the substrate thatintersects the first inlet channel 5401 and second inlet channel 5402 atthe junction 5404. In certain embodiments, the first inlet channel 5401,second inlet channel 5402, and outlet channel 5403 are all formed in asubstrate. Any suitable method or combination of methods for formingchannels in substrates may be used including, but not limited to,mechanical drilling, end-milling, laser-drilling, wire EDM, chemicaletching, photolithography. In preferred embodiments, the surfaces of thetubing and/or substrates, in particular, surfaces which contact flow ofdispersed phase, comprise materials that have a higher affinity for atleast one of the continuous phases than for the partitions of thedispersed phase so as to, e.g., prevent wetting of the surfaces of thetube and/or substrate by the dispersed phase, which could lead to crosscontamination or coalescence of the dispersed phase partitions.

FIG. 55—Y-style separator with off-chip detection FIG. 55 shows anotherarrangement for increasing the average spacing between partitions ofdispersed phase, where the separation occurs in channels in a substrateand the interrogation region is in a tube. The arrangement comprises afirst inlet channel 5501 with an inlet 5502, a second inlet channel 5503with an inlet 5504, an outlet channel 5505, a junction 5506 where thefirst inlet channel 5501 and second inlet channel 5503 meet and exit inthe outlet channel 5505, an outlet tube 5507, an interrogation region5508, and a substrate 5509 in which the first inlet channel 5501, thesecond inlet channel 5503, and the outlet channel 5505 are formed. Afirst continuous phase and partitions of at least one dispersed phaseenter the first inlet channel 5501 through the inlet 5502, and a secondcontinuous phase enters the second inlet channel 5503 in the inlet 5504.The flows in the first inlet channel 5501 and the second inlet channel5503 combine at the junction 5506, where the second continuous phase isadded so as to increase the average spacing of partitions of dispersedphase. The angle at which the first inlet channel 5501 and the secondinlet channel 5503 meet at the junction 5506 may vary. In certainembodiments, the first inlet channel 5501 and second inlet channel 5503may meet at a relatively small angle between their respective axes, asin FIG. 54, such as an angle of less than 90, 80, 70, 60, or 50 degrees.In certain embodiments, the first inlet channel 5501 and second inletchannel 5502 may meet such that they are nearly or completelyorthogonal, as in FIG. 52. In certain embodiments, the cross-sectionalarea of the outlet channel 5505 may be equal to or less than theequivalent cross-sectional area of the partitions of the dispersed phaseat the junction 5506, and the cross-sectional area of the outlet channel5505 remains equal to or less than the equivalent cross-sectional areaof the partitions of the dispersed phase through the interrogationregion 5508 so that the partitions of the dispersed phase do notsubstantially vary in position when projected onto the cross-section ofthe tube 5507. In other embodiments, the cross-sectional area of theoutlet channel 5505 is larger than the equivalent cross-sectional areaof the partitions of the dispersed phase at the junction 5506, but thecross-sectional area of the outlet channel 5505 subsequently decreasesbefore entering the tube 5507 to a value equal to or less than theequivalent cross-sectional area of the partitions of the dispersedphase. In such embodiments, the second continuous phase can besimultaneously added around more than one partition of the dispersedphase, but the partitions of the dispersed phase would stillsubstantially consume the entire cross-section of the tube 5507 at theinterrogation region 5508, reducing variance in optical signal forpartitions of dispersed phase with identical compositions. Suchembodiments may reduce the pressure drop through outlet channel 5505 byonly restricting the cross-sectional area near the interrogation region5508. In certain embodiments, the outlet channel 5505 at the junction5506 has a cross-sectional area that is at least 1×, 1.05×, 1.1×, 1.15×,1.25×, 1.5× and/or not more than 1.05×, 1.1×, 1.15×, 1.25×, 1.5×, or1.75× the equivalent spherical cross-sectional area of the dispersedphase partitions, or any value in between these values. In theseembodiments, the outlet channel 5505 reduces to a cross-sectional areathat is not more than 1×, 0.95×, 0.9×, 0.85×, 0.8×, 0.75×, 0.7×, 0.6×,0.5×, or 0.4×, and/or at least 0.95×, 0.9×, 0.85×, 0.8×, 0.75×, 0.7×,0.6×, 0.5×, 0.4×, or 0.3×, the equivalent cross-section area of thedispersed phase partitions at a point before the transition to the tube5507. In some embodiments, the cross-sectional area of the tube 5507 isequal to or greater than the diameter of the outlet channel 5505 at thepoint where the tube 5507 and outlet channel 5505 meet so as to reducebreak-up of partitions of dispersed phase when passing from the outletchannel 5505 to the tube 5507. While the cross-sectional area of thetube 5507 may be larger than the cross-sectional area of the outletchannel 5505 at the point where the channel and tube meet, it ispreferred to limit the extent to which the cross-sectional areas differso as to limit the formation of recirculation and dead zones in the tube5507, which can broaden the residence time distribution of dispersedphase partitions in the system. In some embodiments, the cross-sectionalarea of the tube 5507 is at least 0%, 1%, 2%, 5%, 10%, 15%, 25%, 50%, or100% larger and/or not more than 1%, 2%, 5%, 10%, 15%, 25%, 50%, 100%,or 150% larger than the cross-sectional area of the outlet channel 5505at the point where the outlet channel 5505 and tube 5507 meet.

The first inlet channel 5501, second inlet channel 5503, and outletchannel 5505 are formed in a substrate 5509. The channels may be formedby any suitable method or combination of methods including, but notlimited to, mechanical drilling, end-milling, laser-drilling, wire EDM,chemical etching, photolithography. In preferred embodiments, thesurface of the substrate 5509, in particular the surfaces that come incontact with dispersed phase, comprises a material that has a higheraffinity for at least one continuous phase than for the at least onedispersed phase so as to prevent wetting of the surface of the substrate5509 by the at least one dispersed phase. In certain embodiments, morethan 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the substrate 5509comprises a material that has a higher affinity for at least onecontinuous phase than or the at least one dispersed phase. In suchembodiments, wear of the surface has a reduced risk of exposing materialthat has a higher affinity for at least one dispersed phase than atleast one continuous phase. In some embodiments, at least one continuousphase comprises a fluorinated oil, at least one dispersed phasecomprises water, and the surface of the substrate 5509 comprises afluoropolymer. In certain embodiments, both the first continuous phaseand the second continuous phase comprise fluorinated oils.

Thus, in some instances, a first channel may comprise a flowing emulsionleaving the thermal cycler meets a second channel comprising additionalcontinuous phase at a v-based junction. At a portion of the junction,the cross-section of the combined flow channel may be substantiallyreduced to a cross-section smaller than the typical cross-section of adroplet (partition) in the system. In some instances, the combined flowchannel is reduced at a 90 degree angle. In some instances, the combinedflow channel is reduced at a 30 degree, 45 degree, 60 degree, 90 degree,120 degree, 150 degree, 180 degree, or 360 degree angle. The continuousphase may be directly incorporated into the flow from the thermalcycler.

FIG. 51—Tube-in-tube separator FIG. 51 shows another system forincreasing the average spacing between partitions of dispersed phase.The system comprises a first inlet conduit 5101 with an inlet 5102 and asecond inlet conduit 5103 with an inlet 5104, where the first inletconduit 5101 is partially or completely surrounded by the second inletconduit 5103. A first continuous phase and partitions of at least onedispersed phase enter the first inlet conduit 5101 at inlet 5102, and asecond continuous phase enters the second inlet conduit 5103 at inlet5104. The first continuous phase and partitions of dispersed phase exitthe first inlet conduit at a junction region 5105, where they arecombined with second continuous phase so as to increase the averagespacing ‘a’ between partitions of dispersed phase in the continuousphases to a value ‘b’, where b>a. “a” and “b” can be, e.g., the distancebetween the geometric centers of adjacent partitions, or other suitablemeasuring point. In certain embodiments, b is at least 102, 105, 110,125, 150, 175, 200, 225, or 300% of a, and/or b is at most 105, 110,125, 150, 175, 200, 225, 300 or 400% of a. Additionally oralternatively, separation of partitions may be any suitable distancefrom one partition to adjacent partition, as described elsewhere herein.The system further comprises an outlet conduit 5106 and an outlet 5107.The outlet conduit 5106 may comprise an interrogation region formeasuring an optical property of the partitions of the at least onedispersed phase. In some embodiments, the cross-sectional area of theoutlet conduit 5105 is equal to or less than the equivalent sphericalcross-sectional area of the partitions of the dispersed phase at theinterrogation region so that the partitions of the dispersed phaseocclude or substantially occlude the entire cross-sectional area of theoutlet conduit.

In certain embodiments, the first inlet conduit 5101 and second inletconduit 5103 are tubular, with the first inlet conduit 5101substantially co-axial with the second inlet conduit 5103. Connectionsto the inlets 5102 and 5104 may be made in any suitable manner, such asthose described for partitioners. The diameter of the first inletconduit 5101 can be sized such that the partitions of the at least onedispersed phase are in single-file or substantially in single-file atthe junction 5105. In certain embodiments, the cross-sectional area ofthe first inlet conduit 5101 at the junction 5105 is not more than 80%,90%, 95%, 100%, 105%, 110%, or 120%, 125%, 150%, or 175% and/or at least70%, 80%, 90%, 95%, 100%, 105%, 110%, or 120%, 125%, or 150% of theequivalent spherical cross-sectional area of the partitions of the atleast one dispersed phase.

In other embodiments, the first inlet conduit 5101, the second inletconduit 5103, and the outlet conduit 5106 are channels formed into asubstrate. In certain embodiments, the inlets 5102 and 5104 are in fluidcommunication with interfaces to the substrate that are orthogonal orsubstantially orthogonal to the direction of flow in the first inletconduit 5101 and second inlet conduit 5103 at the junction of the firstinlet conduit 5101 and second inlet conduit 5103. In certainembodiments, the outlet conduit 5106 is co-planar or substantiallyco-planar with the first inlet conduit 5101 and second inlet conduit5103. In other embodiments, the flow axis of the outlet conduit 5106 isorthogonal or substantially orthogonal to the flow axis of the firstinlet conduit 5101 and the flow axis of the second inlet conduit 5103.In such embodiments, it is preferred that the direction of flow of theoutlet conduit 5103 be oriented relative to gravity to use buoyant forceto improve the achieved separation of the partitions of the at least onedispersed phase. For example, if at least one dispersed phase has a massdensity lower than that of the first continuous phase and the secondcontinuous phase, the direction of flow can be oriented in the directionof decreasing gravitational field strength so that the relative velocityof the partitions of the at least one dispersed phase and the continuousphases increases. The conduits may be formed in the substrate by anysuitable method, as described elsewhere herein for use with substrates.In certain embodiments, the outlet conduit 5106 comprises aninterrogation region. In other embodiments, another conduit interfacesto the outlet conduit 5106 at outlet 5107, and that conduit comprises aninterrogation region for detecting at least one optical property of thepartitions of the at least one dispersed phase.

The first inlet conduit 5101, second inlet conduit 5103, and outletconduit 5105 may be constructed of any suitable material. In someembodiments, surfaces of the first inlet conduit 5101, second inletconduit 5103, and outlet conduit 5105, in particular surfaces thatencounter flow of dispersed phase, comprise materials that have a higheraffinity for at least one continuous phase than for at least onedispersed phase so as to reduce the likelihood of wetting of thesurfaces by the at least one dispersed phase. Continuous phase can beany continuous phase as described herein; in certain embodiments thecontinuous phase comprises a fluorinated oil, in some cases including asurfactant, such as a fluorosurfactant; fluorinated oils andfluorosurfactants are as described elsewhere herein.

Thus, an exemplary system for adding a continuous phase to separate eachdroplet (partition) may comprise a microfluidic channel or tube, e.g.,from a reactor enters a junction where it becomes substantiallyconcentric with a larger microfluidic tube or channel. Continuous phasemay be added in the annular space between the larger microfluidic tubeor channel and the smaller microfluidic tube or channel. At a point inthe junction, the inner microfluidic tube or channel terminates, and theemulsion flow in that channel joins the additional continuous phaseflowing in the larger microfluidic tube or channel. A converging sectionbeyond the junction can bring the inner diameter of the droplet(partition) flow channel down to that required for the optical stage.

FIGS. 56A and 56B—Constricted tube separator. FIGS. 56A and 56B showdifferent views of a system that may increase the separation betweenpartitions of dispersed phase in a flow of a first continuous phase thatdoes not require the addition of a volume of a second continuous phase.The system comprises a conduit 5601 that comprises an inlet 5602, anoutlet 5603, and a region 5604 where the cross-sectional area of theconduit is smaller than at the inlet 5602 or the outlet 5603. At leastone continuous phase and partitions of at least one dispersed phaseenter the conduit at the inlet 5602. In the region 5604, thecross-sectional area is reduced to a value smaller than the equivalentspherical cross-sectional area of the at least one dispersed phasepartitions, causing them to extrude longitudinally as they fill orsubstantially fill the cross-sectional area of the conduit. As thepartitions of the at least one dispersed phase extrude, they displacefluid comprising the at least one continuous phase into the regionsbetween the partitions of the at least one dispersed phase, increasingthe distance (as measured along the conduit main axis) between thepartitions of the at least one dispersed phase. In the region 5604, thecross-sectional area of the conduit may be not more than 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 95% and/or at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% of the equivalent spherical cross-sectional areaof the partitions of the dispersed phase. This increases both separationof partitions and the overall length of partitions in a manner and to adegree that can be calculated.

In certain embodiments, the conduit 5601 comprises the internal volumeof a length of tubing. In these embodiments, the conduit 5601 may beformed in the tubing by any suitable method. In other embodiments, theconduit 5602 may be formed as a channel in a substrate. Some suchembodiments are shown in FIGS. 57A and 57B. FIG. 57A shows an embodimentwhere the conduit 5701 is formed in a rectangular substrate. FIG. 57Bshows an embodiment where the conduit 5701 is formed in a monolith, inthis Figure depicted as a cylindrical monolith. The channel may beformed by any suitable method, including mechanical drilling, laserdrilling, surface milling, end-milling, etching, photolithography. Incertain embodiments, the surface of the conduit 5701 comprises amaterial that a higher affinity for the at least one continuous phasethan for at least one dispersed phase, such that the likelihood that theat least one dispersed phase will wet the surface of the conduit 5701 isreduced.

FIG. 58—Typical interrogation region. A system for interrogating atleast one optical property of partitions in a dispersed phase flowing inan emulsion with at least one continuous phase is shown in FIG. 58. Thesystem comprises a conduit 5801, and inlet 5802, an outlet 5803, and aninterrogation region 5804. In certain embodiments, the cross-sectionalarea of the conduit 5801 in the interrogation region 5804 is smallerthan the cross-sectional area of the conduit 5801 at the inlet 5801 orthe outlet 5803. For example, the cross-sectional area of the conduit5801 in the interrogation region 5804 can be equal to or less than theequivalent spherical cross sectional area of the partitions of thedispersed phase such that the partitions of the dispersed phase fill orsubstantially fill the cross-sectional area of the conduit 5801. Thisreduces the variability of measurements of optical properties of thepartitions of the dispersed phase due to variation in the position ofthe partitions of the dispersed phase as projected onto thecross-sectional area of the conduit 5801 in the interrogation region5804. It will be appreciated that, as described for FIG. 56, such anarrowing both elongates partitions (if cross-sectional area of theconduit is less than that of partitions) and increases separationbetween partitions (because the same volume of continuous phase must fitinto a smaller conduit, thus length must increase). In certainembodiments, in the region 5804, the cross-sectional area of the conduitmay be not more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%and/or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of theequivalent spherical cross-sectional area of the partitions of thedispersed phase. The system additionally comprises at least oneexcitation source 5805, one excitation lens 5806, one photodetectionelement 5807 and one emission lens 5808. The excitation lens 5804 has afirst focal length and is positioned so that a first wavelength ofelectromagnetic radiation emitted by the excitation source 5805 isfocused or substantially focused on the center of the conduit 5801 at apoint in the interrogation region 5804. The emission lens 5804 has asecond focal length and is positioned so that a second wavelength ofelectromagnetic radiation emitted by a first component comprising thepartitions of dispersed phase is collimated as it passes in thedirection of the photodetection element 5807. At least one continuousphase and partitions of dispersed phase flow into the inlet 5802 andpass into the interrogation region 5804. The first wavelength ofelectromagnetic radiation emitted by the excitation source 5805 isfocused on the interrogation region 5804, where it may excite at leastone component of one or more of the partitions of dispersed phaseflowing through the interrogation region. This at least one componentthen emits electromagnetic radiation in the second wavelength thatpasses through the emission lens 5808, where it is collimated heading tothe detection element 5807. Collimated electromagnetic radiationimpinging on the excitation lens 5806 has a first axis parallel to thedirection of collimation, and collimated electromagnetic radiationleaving the emission lens 5808 has a second axis parallel to thedirection of collimation. In general, the first and second axes may haveany relative angle. In certain embodiments, the first and second axisform an orthogonal or substantially orthogonal angle so thatelectromagnetic radiation emitted by the excitation source 5805 incidenton the detection element is reduced relative to embodiments where thefirst and second axis are not orthogonal or substantially orthogonal.Thus, in certain embodiments, the angle between the first and secondaxes is between 45 degrees and 135 degrees, between 60 degrees and 120degrees, between 75 degrees and 105 degrees, between 85 degrees and 95degrees, or between 89 degrees and 91 degrees.

In certain embodiments, the conduit 5801 comprises the internal volumeof a tube. Such embodiments have the advantage that the electromagneticradiation impinging on the outer surface of the tube is normal to theouter surface of the tube at any position on a plane normal to thecentral axis of the conduit 5801, reducing the reflection ofelectromagnetic radiation from the outer surface of the tube. In otherembodiments, the conduit 5801 comprises a channel in a substrate.

FIG. 59—Tubular interrogation region with opposing excitation/detection.FIG. 59 shows a further embodiment of the system shown in FIG. 58. Thesystem comprises a conduit 5901, an inlet 5902, an outlet 5903, aninterrogation region 5904, an excitation source 5905, an excitationfilter 5906, an excitation lens 5907, an emission lens 5908, an emissionfilter 5909, an optical restriction 5910, and a detection element 5911.Electromagnetic radiation in a first wavelength range A is emitted bythe excitation source 5905. In certain embodiments, the excitationsource 5905 comprises an optical element to collimate theelectromagnetic radiation in a first wavelength range A. Theelectromagnetic radiation passes through the excitation filter 5906where electromagnetic radiation outside of a second wavelength range A′is reduced in intensity. In preferred embodiments, the second wavelengthrange A′ coincides or substantially coincides with wavelengths where atleast one component of one or more of the partitions of dispersed phaseis excited so as to emit electromagnetic radiation in a third wavelengthrange B. After passing through the excitation filter 5906, theelectromagnetic radiation passes through an excitation lens 5907 whereit is focused on the conduit 5901 in the interrogation region 5904 andmay excite at least one component of one or more of the partitions ofdispersed phase in the interrogation region 5904 such that the componentemits electromagnetic radiation in the third wavelength range B andpasses into the emission lens 5907 where it is collimated onto theemission filter 5909. The emission filter 5909 is chosen so that itreduces the radiant power of electromagnetic radiation in the thirdwavelength range B outside of a fourth wavelength range B′. In certainembodiments, the fourth wavelength range B′ is a subset of the thirdwavelength range B. The optical restriction 5910 is located between theemission filter 5909 and the detection element 5911 such that itrestricts the field of view of the detection element so that theelectromagnetic radiation reaching the detection element is limited orsubstantially limited to that emitted by at least one component in asingle partition of the dispersed phase. This allows, in a practicalmanner, for the measurement of an optical property of only one partitionof the dispersed phase at any specific time. In certain embodiments, atleast 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%and/or not more than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%,98%, 99% or 100% of the electromagnetic radiation reaching the detectionelement was emitted by at least one component in a single partition ofthe dispersed phase. In certain embodiments, when a first partition ispassing through the interrogation zone, not more than 0.1%, 0.5%, 1%,2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% and/or at least0, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, or 40%of the electromagnetic radiation reaching the detection element due toone or more components in a partition is from one or more partitionsother than the partition in the interrogation zone. Any suitablegeometry may be used for the optical restriction. In certainembodiments, the optical restriction is a pinhole, i.e., a circularaperture. The pinhole can be any suitable size, for example, with adiameter less than 1000 um, 750 um, 500 um, 250 um, 150 um, 100 um, 80um, or 50 um and/or at least 750 um, 500 um, 250 um, 150 um, 100 um, 80um, 50 um, or 25 um. In other embodiments, the optical restriction 5910is a slot or a star shape. In some embodiments, the emission filter 5909additionally comprises a system for focusing the electromagneticradiation onto the optical restriction 5910.

FIG. 60—Tubular interrogation region with in-path excitation/detection.FIG. 60 shows a system for measuring at least one optical property ofthe partitions of a dispersed phase where excitation electromagneticradiation and emission electromagnetic radiation share a common opticalpathway. The system comprises a conduit 6001, which comprises an inlet6002, an outlet 6003, and an interrogation region 6004. In certainembodiments, the cross-sectional area of the interrogation region 6004is reduced relative to the inlet 6002 and/or the outlet 6003 such thatit has a value equal to or less than the equivalent sphericalcross-sectional area of the partitions of the dispersed phase. In suchembodiments, the partitions of the dispersed phase fill or substantiallyfill the cross-section of the conduit 6001 in the interrogation region6004, reducing variability in the measurement of an optical property ofthe partitions of the dispersed phase due to variation in the positionof the partitions of the dispersed phase in the cross-section of theconduit 6001. At least one continuous phase and partitions of at leastone dispersed phase enter the conduit 6001 at the inlet 6002 and flowtoward the outlet 6003. In certain embodiments, the partitions of the atleast one dispersed phase enter the inlet 6002 in single file. Incertain embodiments, the partitions of the at least one dispersed phaseare separated along the axis of the conduit 6001 by a volume of the atleast one continuous phase.

The system additionally comprises an excitation source 6005, anexcitation collimator 6006, an excitation filter 6007, a turning mirror6008, an interrogation region lens 6009, an emission filter 6011, anoptical restriction 6012, a detection element 6013, and a collectiontube 6014. The system may be used to detect at least one opticalproperty of the partitions of the at least one dispersed phase asfollows. Electromagnetic radiation with wavelengths in a firstwavelength range A is emitted by the excitation source 6005 andcollimated by the excitation collimator 6006. In certain embodiments,the collimator is a lens. In other embodiments, the collimator is aparabolic mirror. The electromagnetic radiation in the first wavelengthrange A passes through the excitation filter 6007, which reduces thepower of the electromagnetic radiation outside of a second wavelengthrange A′. In certain embodiments, the second wavelength range A′ is asubset of the first wavelength range A. In certain embodiments, thesecond wavelength range A′ coincides or substantially coincides with athird wavelength range A″ such that the third wavelength range A″excites a component contained in one or more of the partitions of the atleast one dispersed phase, subsequently causing it to emitelectromagnetic radiation in a fourth wavelength range B. Theelectromagnetic radiation in the second wavelength range A′ transmittedby the excitation filter 6007 is then incident on the turning mirror6008, where it is redirected at an angle alpha away from an axis normalto the surface of the excitation filter 6007. In certain embodiments,the angle alpha is between 85 degrees and 95 degrees. The turning mirrorcan be any suitable mirror, e.g., a dichroic mirror. Electromagneticradiation in the wavelength range A′ reflected from the turning mirrorpasses through an interrogation region lens 6009, where it is focused onthe interrogation region. In certain embodiments, the focal length ofthe interrogation region lens 6009 is selected and the lens 6009positioned such that the electromagnetic radiation in the wavelengthrange A′ is focused or substantially focused on the center of theconduit 6001 in the interrogation region 6004. Electromagnetic radiationin the wavelength range A′ excites a component contained in one or morepartitions of the at least one dispersed phase, causing it to emitelectromagnetic radiation in the fourth wavelength range B. Theelectromagnetic radiation in the fourth wavelength range B is collimatedby the interrogation region lens 6009 and is then incident on theturning mirror 6008 and is substantially transmitted by the turningmirror 6008. The electromagnetic radiation in the fourth wavelengthrange B is then incident on the emission filter 6011, which reduces theradiant power outside of a fifth wavelength range B′. In someembodiments, the fifth wavelength range B′ coincides with all or asubstantial portion of the fourth wavelength range B so as to maximizetransmitted power. The emission filter 6011 can further comprise afocusing optic that concentrates the electromagnetic radiation in thefifth wavelength range B′ on the optical restriction 6012. The opticalrestriction 6012 is sized and positioned so that the field of view ofthe detection element 6013 is substantially restricted toelectromagnetic radiation arriving from only at most a single partitionof the at least one dispersed phase. In certain embodiments, at least30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% and/or notmore than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% of the electromagnetic radiation reaching the detection element wasemitted by at least one component in a single partition of the dispersedphase. In certain embodiments, when a first partition is passing throughthe interrogation zone, not more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%,7%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% and/or at least 0, 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, or 40% of theelectromagnetic radiation reaching the detection element due to one ormore components in a partition is from one or more partitions other thanthe partition in the interrogation zone. As such, little or nocorrection in partition intensity must be made to account for the atleast one emitting component comprising partitions of the dispersedphase preceding or following the partition of the dispersed phaseemitting a plurality of electromagnetic radiation reaching the detectionelement.

In some embodiments, the conduit 6001 comprises the internal volume of atube comprising a material that is at least partially transmissive inthe third wavelength range A″ and the fourth wavelength range B. Incertain embodiments, the tube comprises a material at least partiallytransmissive in the third wavelength range A″ and the fourth wavelengthrange B, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%and/or not more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%transmissive of electromagnetic radiation of wavelength range A″ andleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% and/or not morethan 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% transmissive ofelectromagnetic radiation of wavelength range B. In certain embodiments,the tube transmits at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 75%, at least 85%, at least90% or at least 95% of the electromagnetic radiation incident upon it.In other embodiments, the conduit 6001 comprises a channel in asubstrate. In some embodiments, the substrate comprises a material atleast partially transmissive in the third wavelength range A″ and thefourth wavelength range B such as at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 99% and/or not more than 30%, 40%, 50%, 60%, 70%, 80%, 90%,99%, or 100% transmissive of electromagnetic radiation of wavelengthrange A″ and least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% and/ornot more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%transmissive of electromagnetic radiation of wavelength range B. Incertain embodiments the substrate transmits at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 75%, at least 85%, atleast 90%, or at least 95% of the electromagnetic radiation incidentupon it.

FIG. 61—On-chip interrogation region with opposing excitation/detection.FIG. 61 shows a system for detecting an optical property of partitionsof a dispersed phase flowing in a channel. The system comprises asubstrate 6101, a flow channel 6102, an inlet 6103, an outlet 6104, anexcitation source 6105, an excitation lens 6106, an emission lens 6107,a detection element 6108, and an interrogation region 6109. The flowchannel 6102 is formed in the substrate 6101. At least one continuousphase and partitions of at least one dispersed phase enter the flowchannel 6102 at the inlet 6103 and flow toward the outlet 6104. In someembodiments, the flow channel 6102 is narrowed in the interrogationregion 6109 so that the cross-sectional area of the flow channel 6102 inthe interrogation region 6109 is less than or equal to the equivalentspherical cross-sectional area of the partitions of the dispersed phase.

The excitation source 6105 emits substantially collimatedelectromagnetic radiation in a first wavelength range A in the directionof the excitation lens 6106. The excitation lens 6106 focuseselectromagnetic radiation in the first wavelength range A on theinterrogation region 6104 of the flow channel 6102, where it excites atleast one component of at least one of the partitions of the at leastone dispersed phase, causing it to emit electromagnetic radiation in asecond wavelength range B. The substrate 6101 possesses a firsttransmissivity in the wavelength range A and a second transmissivity inthe second wavelength range B such that the substrate 6101 transmits asubstantial fraction of incident electromagnetic radiation in eachwavelength range. In some embodiments, the substrate 6101 transmits atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95% of the incidentelectromagnetic radiation in the first wavelength range A, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95% of the incident electromagneticradiation in the second wavelength range B, or any combination thereof.Electromagnetic radiation emitted in the second wavelength range B istransmitted through the substrate 6101, where it is incident on anemission lens 6107 and is collimated in a first axis.

FIGS. 62A and 62B—Use of an optical restriction to limit acceptedelectromagnetic radiation to a single partition. FIG. 62A shows a systemfor measuring an optical property of partitions of at least onedispersed phase that does not comprise an optical restriction between anemission filter and a photodetection element, and FIG. 62B shows asystem for measuring an optical property of partitions of at least onedispersed phase that does comprise an optical restriction between anemission filter. In the system shown in FIG. 62A, the field of view ofthe interrogation region is not limited or substantially limited to asingle partition. As a result, optical measurements at the detectionelement quantify energy from more than a single partition of the atleast one dispersed phase at any one time, and the signal (as shown inthe accompanying plot of intensity against time) has peaks representingthe central portion of a single partition of the at least one dispersedphase that may be difficult to distinguish from the background signal.In contrast, the system shown in FIG. 62B comprises an opticalrestriction that limits the fraction of electromagnetic radiationreaching the detection element such that all or a substantial fractionof the energy emitted from partitions and reaching the detection elementat any one time is from only a single partition of the at least onedispersed phase. In some embodiments, this fraction is greater than 20%,greater than 30%, greater than 40%, greater than 50%, greater than 60%,greater than 70%, greater than 80%, greater than 90%%, greater than 95%,greater than 98%, or greater than 99%. In certain embodiments, at least30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% and/or notmore than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% of the electromagnetic radiation reaching the detection element wasemitted by at least one component in a single partition of the dispersedphase. In certain embodiments, when a first partition is passing throughthe interrogation zone, not more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%,7%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% and/or at least 0, 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, or 40% of theelectromagnetic radiation reaching the detection element due to one ormore components in a partition is from one or more partitions other thanthe partition in the interrogation zone. In the plot of intensityagainst time accompanying FIG. 62B, the amplitude difference betweenpeak values and trough values is relatively larger than in the opticalrestriction-free system shown in FIG. 62A. This is due to the reducedcontributions to the overall intensity signal from partitions of the atleast one dispersed phase upstream or downstream of the partition of theat least one dispersed phase nearest the system focal point in theinterrogation region. The optical restriction may take any suitableshape provided it reduces the fraction of electromagnetic radiationincident on the detection element to a portion of the energy that wouldotherwise reach it; for example, it can reduce the fraction ofelectromagnetic radiation incident on the detection element not emittedby a component of the partition of the at least one dispersed phase mostproximate to the focal point of the interrogation region. In someembodiments, the optical restriction is circular. In certainembodiments, the optical restriction has a size of 10-500 um (diameterof circular optical restriction), e.g., 30-400 um, such as 50-350 um, incertain embodiments 150-250 um. Other sizes and positioning are asdescribed elsewhere herein. In other embodiments, the opticalrestriction is shaped like a rectangular slot, triangle, star, pentagon,hexagon, square, heptagon, octagon, or any other n-sided polygon.

FIG. 63—Positive signal partition detection. FIG. 63 shows thephotodetector signal as a function of time as partitions of the at leastone dispersed phase pass through an interrogation region and are spacedsuch that there is a volume of a continuous phase between the partitionsof the at least one dispersed phase and no point of the surface of afirst partition of the at least one dispersed phase is in contact withthe surface of any other partition of the at least one dispersed phasein the interrogation region. As partitions of the dispersed phase enterthe interrogation region and a component contained in one or more thepartitions of the at least one dispersed phase is excited byelectromagnetic radiation in a first wavelength range, a signal (e.g.,voltage) measured at the photodetector increases due to electromagneticradiation emitted in a second wavelength by the component contained inone or more the partitions of the at least one dispersed phase. Thesignal rises until the partition of the at least one dispersed phase iscentered in the interrogation region, after which the signal falls untilthe partition of the at least one dispersed phase is no longer in theinterrogation region. Each peak in signal corresponds to a singlepartition of the at least one dispersed phase. The magnitude of thesignal corresponds to a property of the excited component in thepartitions of the at least one dispersed phase. In certain embodiments,the signal increases with increasing concentration of an optical dye,such as described elsewhere herein. In certain embodiments, theconcentration of the optical dye can be related to the concentration ofa second component in the at least one dispersed phase. The secondcomponent can be any suitable component, e.g., a nucleic acid, protein,molecule, atomic species, solid particulate species, or ion. In somecases, it is desired only to determine which, of a discrete set of valuebins, the concentration of the second component is in, and so signalsmay be classified into “high”, “low”, or any series of discrete values.In some cases, the assay is a digital assay where the concentration ofthe second component may be determined by estimating a statisticaldistribution from the number of “high” and “low” concentrations measuredfor the second component contained in one or more of the partitions ofthe dispersed phase. In certain embodiments, the assay is digitalpolymerase chain reaction and the second component is a nucleic acid.

FIG. 64—Negative signal partition detection. FIG. 64 shows thephotodetector signal as a function of time as partitions of the at leastone dispersed phase pass through an interrogation region and are notspaced so that no point of the surface of a first partition of the atleast one dispersed phase is in contact with the surface of any otherpartition of the at least one dispersed phase in the interrogationregion. In such a case, the signal at the photodetector may compriseelectromagnetic radiation contributions from more than one partition ofthe at least one dispersed phase at a given time and the signal does notfall to a baseline in between signal peaks. Each relative peak stillcorresponds to a point where a single partition of the at least onedispersed phase is centered in the system, and a measurement of thesignal intensity for that partition may be made. In certain embodiments,a correction to the signal for a first peak representing a firstpartition is made by accounting for the value of the signals at thepeaks of the partitions following and preceding the first peakrepresenting the first partition. In a certain embodiments, theintensity of the first peak is reduced by a first coefficient multipliedby the value of the signal of the peak immediately preceding the firstpeak in time and reduced by a second coefficient multiplied by the valueof the signal of the peak immediately following the first peak. Incertain embodiments, the first and second coefficient are proportionalto the size and position of an optical restriction relative to theinterrogation region. In a further embodiment, the first and secondcoefficient have the same value.

FIG. 65A—Multi-excitation source in tubular arrangement; FIG. 65B. FIG.65 shows systems for measuring an optical property of one or morepartitions of at least one dispersed phase flowing in at least onecontinuous phase where the flow is in a tubular conduit. The systemcomprises a tubular conduit with an outer surface 6501 and an innersurface 6502. A first excitation source 6503 emits electromagneticradiation in a first wavelength range A that is collimated by a firstexcitation collimating lens 6504 onto a first excitation filter 6505,which substantially reduces the radiant power transmitted through thefilter outside of a wavelength range A′. In some embodiments, thefraction of incident radiant power outside of the wavelength range A′transmitted by the filter is less than 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵,10⁻⁶, 10⁻⁷, or 10⁻⁸. A first excitation focusing lens 6506 focuses theradiant power in the wavelength range A′ onto a focal point in or nearthe tubular conduit. The tubular conduit has a wall constructed of amaterial that at least partially transmits the radiant power in thewavelength range A′ focused by the first focal lens and has a distancebetween the outer surface 6501 and the inner surface 6502 such that thetotal reduction in radiant power is small. In some embodiments, thetotal reduction is radiant power from the outer surface 6501 to theinner surface 6502 is less than 75%, 65%, 55%, 50%, 40%, 30%, 20%, 10%,5%, 1%, 0.1%, or 0.01% and/or at least 65%, 55%, 50%, 40%, 30%, 20%,10%, 5%, 1%, 0.1%, 0.01%, 0.001%, or 0%. In some embodiments, thefocusing lens 6506 is positioned such that the collimatedelectromagnetic radiation in the wavelength range A′ has a first axisnormal or substantially normal to the outer surface 6501, which mayminimize reflection of the electromagnetic radiation in the wavelengthrange A′ off of the outer surface 6501. The electromagnetic radiation inthe wavelength range A′ excites at least one component in one or more ofthe partitions of the at least one dispersed phase, causing it to emitelectromagnetic radiation in a wavelength range B. The system comprisesan aperture 6516 that limits the field of view of a region of the systemto an angle alpha in the plane normal to the flow axis of the conduit.Doing so may reject some, most, or substantially all of theelectromagnetic radiation in the wavelength range A′ emitted by thefirst excitation source 6503 and filtered by the first excitation filter6505 reaching an emission collimating lens 6515 and any subsequentphotodetection element beyond the lens 6515.

In certain embodiments, the aperture 6516 is folded such it increases,e.g., maximizes the angular extent available to excitation sourceswithout allowing substantial electromagnetic radiation from theexcitation sources to pass through the aperture, as shown in FIG. 65B.The system additionally comprises a detection element. The detectionelement may be any suitable element. In certain embodiments, thedetection element is one of a photodiode, silicon photomultiplier(SiPM), avalanche photodiode, complementary metal oxide semiconductor(CMOS) detector, charge-coupled device (CCD) camera, or photomultipliertube (PMT). The detection element can be positioned so as to measure anintensity or a power of electromagnetic radiation passing through theaperture 6516. In certain embodiments, the detection element ispositioned so as to measure an intensity or a power of electromagneticradiation passing through collimation lens 6515. In further embodiments,the system additionally comprises one or more of an emission filter, anemission focusing lens, and emission optical restriction. The emissionfilter is configured to substantially reduce the electromagneticradiation passed outside at least one wavelength range B′. In preferredembodiments, the wavelength range B′ coincides or substantiallycoincides with the wavelength range B. The emission focusing lensfocuses electromagnetic radiation onto the emission optical restriction,restricting the field of view, for example, restricting the field ofview such that the electromagnetic radiation reaching the detectionelement originated primarily from a single partition of the at least onedispersed phase.

In certain embodiments, the system further comprises a second excitationsource 6507 that emits electromagnetic radiation in a wavelength rangeC, a second excitation collimating lens 6508, a second excitation filter6509 that reduces the radiant power transmitted through the filteroutside of a wavelength range C′, and a second excitation focusing lens6510 that focuses the electromagnetic radiation in the wavelength rangeC′ on partitions of the at least one dispersed phase flowing through theconduit. The electromagnetic radiation in the wavelength range C′ mayexcite at least one component contained in one or more of the partitionsof the at least one dispersed phase to emit electromagnetic radiation ina wavelength range D. In further embodiments, the system comprises athird excitation source 6511 that emits electromagnetic radiation in awavelength range E, a third excitation collimating lens 6512, a thirdexcitation filter 6513 that reduces the radiant power transmittedthrough the filter outside of a wavelength range E′, and a thirdexcitation focusing lens 6514 that focuses the electromagnetic radiationin the wavelength range E′ on partitions of the at least one dispersedphase flowing through the conduit. The electromagnetic radiation in thewavelength range E′ may excite at least one component contained in oneor more of the partitions of the at least one dispersed phase to emitelectromagnetic radiation in a wavelength range F. In general,embodiments may be extended to comprise four, five, six, seven, eight,or more sets of an excitation source, an excitation collimating lens, anexcitation filter, and an excitation focusing lens that exposepartitions of the at least one dispersed phase flowing through theconduit to electromagnetic radiation in a wavelength range specific toeach excitation set that may excite at least one component contained inon or more of the partitions of the at least one dispersed phase. Eachexcitation wavelength range corresponds to a component contained in oneor more of the partitions of the at least one dispersed phase such thatthe radiant power emitted by each component when excited by eachexcitation wavelength corresponds to a property of the component in thepartitions of the dispersed phase. In certain embodiments, the propertyis a molar or mass concentration of each component in the partitions ofthe dispersed phase. In certain embodiments, the property of eachcomponent in the partitions of the dispersed phase may be related toanother property of the partitions of the dispersed phase. In anexample, the components excited by the electromagnetic radiation in theexcitation wavelength ranges are fluorescent dyes, the radiant poweremitted by the fluorescent dyes may be quantitatively related to theirmolar concentration, and the molar concentration of each fluorescentdye, or a combination of the molar concentrations of a plurality offluorescent dyes, may be related to a molar or mass concentration of atleast one more component of the system. In certain embodiments, such acomponent would be a nucleic acid, a protein, a molecule, an atomicspecies, a solid particulate species, or an ionic species. Byassociating an excitation wavelength range with at least one componentof the partitions of the dispersed phase, the use of at least twoexcitation wavelength ranges can allow the measurement of at least twoproperties of components contained in one or more of the partitions ofthe dispersed phase.

In some embodiments, the photodetection element produces a signalproportional to the incident energy across a wavelength range Z, wherethe wavelength range Z at least partially coincides with at least two ofthe wavelength ranges B′, D′, F′, or more, depending on the number ofexcitation sources. In such cases, multiplexing measurements of at leasttwo components in the partitions of the at least one dispersed phase canrequire methods for deconvoluting signals at the single photodetectionelement. In certain embodiments, the excitation sources are cycledbetween active and inactive states such that, at any given time, amaximum of one excitation source is active. By cycling through theexcitation sources at a rate at least equal to 1/(n*t), where n is thenumber of excitation sources and t is the time required for a singlepartition of the at least one dispersed phase to pass through the fieldof view of the detection element, at least one measurement may be madeat the detection element for each component of the partitions of the atleast one dispersed phase that may be associated with at least oneexcitation source. In preferred embodiments, the cycling frequency is1/(n*t*g), where g is a coefficient equaling the number of measurementsto be made, on average, for the duration of each excitation source,where g is greater than or equal to 4. In some preferred embodiments, gis greater than 4, 5, 6, 8, 10, 20, or 100. The limit to g is related tothe response time of both the photodetection element and the at leastone excited component in the partitions of the at least one dispersedphase. A schematic representation of this is shown in FIG. 66.

In certain embodiment, lock-in amplification is used to deconvolute thesignals from the at least one component, for example at least twocomponents, excited by the at least two wavelengths of electromagneticradiation transmitted by the at least two excitation filters, increasethe signal-to-noise ratio of the detection element, or both. The firstexcitation source 6503 is modulated in a first periodic function, whichtranslates to a modulation in the electromagnetic radiation passed bythe first excitation filter 6505 by the first periodic function and amodulated response of the electromagnetic radiation emitted by a firstcomponent of the partitions of the at least one dispersed phase in thewavelength range B. When represented as a Fourier series, the firstperiodic function will have an infinite set of coefficients, one foreach frequency term in the Fourier series. In certain embodiments, therelative magnitude of each of a finite subset of the coefficients is atleast greater than any other coefficient not in the finite subset of thecoefficients by a minimum multiple. In certain embodiments, the minimummultiple is greater than 10, greater than 100, greater than 1000,greater than 10,000, greater than 100,000, or greater than 1,000,000. Toimprove analysis, it is desired that the finite subset has a limitednumber of coefficients. In some embodiments, the number of coefficientsis fewer than 1000, fewer than 100, fewer than 10, fewer than 5, fewerthan 2, or 1. In certain embodiments, the first periodic function issinusoidal and the number of coefficients in the finite subset is 1. Dueto the orthonormality of sinusoidal functions and the mathematicalcompleteness of the Fourier series, integration of the product of thefirst periodic function and the signal measured by the detection elementover multiple periods of the first periodic function will result inlower measured amplitudes of components of the signal corresponding toterms of the Fourier series representation of the first periodicfunction whose coefficients are not in the finite subset and relativeamplification of the terms of the Fourier series representation of thefirst periodic function whose coefficients are in the finite subset. Ifthe relative magnitude of the coefficients of the Fourier seriesrepresentation of the components of the signal measured by the detectionelement that are not related to the electromagnetic radiation in thewavelength range B in the finite subset are not relatively larger thanthe coefficients of the Fourier series representation of the componentsof the signal measured by the detection element that are not related tothe electromagnetic radiation in the wavelength range B not in thefinite subset, components of the signal measured by the detectionelement related to the electromagnetic radiation in the wavelength rangeB will be preferentially amplified to components of the signal measuredby the detection element not related to the electromagnetic radiation inthe wavelength range B. As such, signals measured by the detectionelement that may be related to a property of at least one component ofthe partitions of the dispersed phase may be amplified by this method tohave a multiple of their signal-to-noise ratio over signals measuredwhen this method is not applied. In certain embodiments, the multiple isgreater than 2, greater than 5, greater than 10, greater than 100,greater than 1000, greater than 10⁵, or greater than 10⁶.

In certain embodiments, the first periodic function is chosen such thatit preferentially avoids terms of the Fourier series representation ofthe components of the signal measured by the detection element that arenot related to the electromagnetic radiation in the wavelength range Bwhose coefficients are relatively larger than other coefficients in thewavelength range B. For example, in many electronics systems, noise hasa power spectrum with higher coefficient frequencies at lowerfrequencies. In certain embodiments, these are frequencies less than 10Hz, less than 100 Hz, less than 1 kHz, less than 10 kHz, less than 20kHz, or less than 100 kHz. By selecting a first periodic function suchthat the coefficients of the terms of the Fourier series representationof the first periodic function representing these frequencies are not inthe finite subset, this low frequency electronic noise may bepreferentially rejected in the integrated measurement. For example, thefirst periodic function may be a sinusoidal function with a frequency of1 MHz and the terms of the Fourier series representation of the signalmeasured by the detection element that are not related to theelectromagnetic radiation in the wavelength range B representingfrequencies greater than 100 kHz are not in the finite subset.

In applying the method to obtain an amplified signal from the signalmeasured by the detection element, the signal measured by the detectionelement typically must be integrated over multiple periods of the firstperiodic function. In general, the signal-to-noise ratio achievedincreases as the number of periods over which the signal measured by thedetection element is integrated increases. However, increasing thenumber of periods increases the time constant for response of theamplified signal (or equivalently, reduces the effective rate at whichthe amplified signal may be sampled). As partitions of the at least onedispersed phase move through the interrogation region, the signalmeasured by the detection element rises then falls. If individualpartitions are to be distinguished, a minimum number of measurements ofthe signal of the detection element must be made when each individualpartition of the first dispersed phase is in the interrogation region.At a very minimum, this minimum number is 1. In some embodiments, theminimum number is at least 3, at least 5, at least 7, at least 8, atleast 10, or at least 20. In preferred embodiments, the minimum numberis at least 10, at least 20, at least 50, or at least 100. This minimumnumber of measurements practically limits, in conjunction with eachother, the number of periods over which the signal measured by thedetection element is integrated and the rate at which the partitions ofthe dispersed phase move through the interrogation region. To increasethe rate at which the partitions of the dispersed phase move through theinterrogation region while maintaining the minimum number ofmeasurements of the signal of the detection element made when eachindividual partition of the first dispersed phase is in theinterrogation region, the number of periods over which the signalmeasured by the detection element is integrated must be decreased,relatively reducing the effective signal-to-noise ratio. Conversely, toincrease the signal-to-noise ratio while maintaining the minimum numberof measurements of the signal of the detection element, the rate atwhich partitions of the dispersed phase move through the interrogationregion must be decreased. In an embodiment, a rate at which partitionsof the dispersed phase move through the interrogation region is at least10,000 partitions per minute and the rate at which measurements of thesignal of the detection element are made is at least 40,000 times perminute.

The method also allows for the simultaneous measurement of properties ofmultiple components of the partitions of the at least one dispersedphase, where the properties multiple components of the partitions of theat least one dispersed phase may be related to electromagnetic radiationin multiple wavelength ranges emitted by the multiple components of thepartitions of the at least one dispersed phase as a result of beingirradiated by electromagnetic radiation in multiple wavelength rangesemitted by a plurality of excitation sources, each modulated in time bya distinct periodic function.

The planar arrangement shown in FIGS. 65A and 65B allows for thepositioning of multiple excitation sources around a single interrogationregion where the direction of the collimating electromagnetic radiationleaving each excitation collimating lens is normal or substantiallynormal to the outer wall 6501. This effectively allows for a high levelof measurement multiplexing for a single interrogation region, reducingcost and allowing for multiple measurements to be made on each partitionof the at least one dispersed phase as it passes through the singleinterrogation region.

In certain embodiments, the excitation sources may be electromagneticradiation emitting diodes, organic electromagnetic radiation emittingdiodes, arrays of electromagnetic radiation emitting diodes, lasers, orany combination thereof. Some excitation sources, such aselectromagnetic radiation emitting diodes, have very low costs. Othersources, such as lasers, have higher costs but effectively smallerwavelength ranges of emission.

FIGS. 67A, 67B, and 67C—Tubular spectrometer arrangements withdiffraction gratings/CCD. FIGS. 67A, 67B, and 67C show furtherembodiments of the system represented in FIG. 65 that allows formultiplexing by spatially resolving the spectral content of theelectromagnetic radiation passing through the aperture 6716. The lens6715 collimates the electromagnetic radiation passing through theaperture 6716. The system further comprises an emission filter 6718 thatreduces the electromagnetic radiation passing through the aperture 6716outside of a wavelength range H. The system further comprises adiffraction grating 6717; the electromagnetic radiation passing throughthe aperture is incident on the diffraction grating 6717, whichdisperses the electromagnetic radiation in a range of angles accordingto a monotonic function of the wavelength of the electromagneticradiation. A solid angle projected from the surface of the diffractiongrating 6717 at an angle alpha will subtend a portion of theelectromagnetic radiation dispersed by the diffraction grating 6717,with the portion representing a fraction of the overall wavelength rangeH. The magnitude of the fraction is monotonically related to the size ofthe solid angle, with larger solid angles covering larger fractions ofthe overall wavelength range H. The system comprises at least onedetection element 6718 that subtends a first solid angle covering awavelength range H′. The wavelength range H′ is selected so that theelectromagnetic radiation in the wavelength range H′ is primarilyresultant from the excitation of a single component of the partitions ofthe at least one dispersed phase. In some embodiments, at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, at least 90%, or atleast 95% of the electromagnetic radiation in the wavelength range H′ isprimarily resultant from the excitation of a single component of thepartitions of the at least one dispersed phase. The concept may beextended so that the system may comprise a second detection elementsubtending a second solid angle intercepting a second wavelength rangeH″, in some cases, a third detection element subtending a third solidangle intercepting a third wavelength range H′″, and so on, with eachwavelength range containing electromagnetic radiation primarilyresultant from the excitation of a single component of the partitions ofthe at least one dispersed phase. Any suitable detection element orcombination of detection elements may be used. In certain embodiments,the detection elements may be a charge-coupled device (CCD) array, a setof photomultipliers, a set of photodiodes, a set of avalanchephotodiodes, a set of silicon photomultipliers, or any combinationthereof.

FIG. 67A shows an embodiment where the diffraction grating 6517 istransmissive, and FIG. 67B shows an embodiment where the diffractiongrating is reflective. FIG. 67C shows the system that further comprisesan optical restriction 6719, such that the electromagnetic radiationincident upon the diffraction grating 6717 results primarily fromemission by components of the partitions of the at least one dispersedphase in a single partition of the at least one dispersed phase. In anembodiment, the fraction is greater than 50%, greater than 60%, greaterthan 70%, greater than 80%, greater than 90%, greater than 95%, greaterthan 98%, or greater than 99%.

FIGS. 68A and 68B—Tubular spectrometer with SiPM detectors. FIGS. 68Aand 68B show further embodiments that additionally comprises at leastone focusing lens 6820 that focuses electromagnetic radiation on atleast one detection element 6818. In FIG. 68A, the diffraction grating6817 is reflective, and in FIG. 68B the diffraction grating istransmissive.

FIG. 69 shows another further embodiment of the system in FIG. 65 formultiplexing. The system additionally comprises at least one turningmirror 6917. The turning mirror preferentially reflects a subset V ofthe wavelength range of electromagnetic radiation passing through theaperture 6916, where the subset V represents electromagnetic radiationprimarily emitted by only a single component of the partitions of the atleast one dispersed phase. A first detection element 6920 measures theintensity or power electromagnetic radiation in the subset V, whereas asecond detection element 6927 measures the intensity or power of theelectromagnetic radiation not in the subset V. The system mayadditionally comprise one or more additional turning mirrors, eachturning mirror preferentially reflecting a different subset of theelectromagnetic radiation passing through the aperture 6916 andcorresponding to electromagnetic radiation primarily emitted by a singlecomponent of the partitions of the at least one dispersed phase. Incertain embodiments, the turning mirror is a dichroic mirror. In certainembodiments, the system comprises an emission filter 6921 that reducesthe power or intensity of the electromagnetic radiation passing throughthe aperture 6916 outside of a wavelength range Z, where Z substantiallycoincides with the wavelengths of electromagnetic radiation emitted bythe components of the partitions of the at least one dispersed phase.

FIG. 70—Offset excitation sources. FIG. 70 shows an embodiment of anexcitation system for a tubular interrogation region where one or moreexcitation sources are offset from a plane normal to a central axis ofthe interrogation region. The system comprises a conduit 7001 thatcomprises an inlet 7002 and an outlet 7003 and an interrogation region7004. Partitions of at least one dispersed phase flow from the inlet7002 through the interrogation region 7004 toward the outlet. Theconduit 7001 comprises an axis perpendicular to the cross section of theconduit in the interrogation region, and a plane co-planar with thecross-section of the conduit and normal to the axis. Excitation sourcesplaced in a co-planar manner with the plane will minimize reflection ofexcitation radiant energy off the outer surface of the conduit 7001, butthere may be a spatial limitation to the number of excitation sourcesthat may be placed in this plane. In the embodiment shown, at least oneexcitation source 7005 is placed so that radiant energy passing throughan excitation collimator 7006, an excitation filter 7007 to restrict thewavelengths of radiant energy passing through the filter to a subsetthat substantially coincides with the ranges of wavelengths forexcitation of at least one component of the partitions, and a focusinglens 7008 to focus light on the interrogation region, where the focusinglens 7008 focuses light at an angle alpha offset from the plane normalto the axis. Doing so may allow for relieving spatial crowding andfitting more excitation sources around the interrogation region, at thepenalty of additional radiant energy loss due to reflection. In someembodiments, alpha is less than 30 degrees, less than 20 degrees, lessthan 15 degrees, or less than 10 degrees.

FIG. 66—Temporal modulation pattern (one LED on at a time). FIG. 66shows a representative diagram of temporal modulation, showing thatexcitation channel 1 is on when excitation channel 2 is off, and viceversa.

FIG. 71—Lock-in detection in partitions. FIG. 71 shows examples ofexciting two radiant energy sources with separate periodic functions,each having a at least partially non-overlapping subset of terms in itsFourier series representation whose coefficients are at least a multiplegreater than the coefficients of all the other terms in their respectiveFourier series representations. The resultant time-domain signal, withthe total signal being the superposition of the contributions from eachmodulated excitation source and random noise, shows distinct peaksaround the main terms of the respective Fourier series expansions ofeach modulated source when transforming to the frequency domain.Selecting each subset of frequency corresponding to each modulatedexcitation source and performing an inverse transform back to the timedomain of that subset, a set of amplified time-domain signalscorresponding primarily to the contribution for each modulatedexcitation source may be obtained.

FIGS. 72A and 72B—Blank sample discrimination. FIG. 72 showsrepresentative signals associated with the discrimination of boundariesbetween samples. In FIG. 72A a first sample comprising a plurality ofpartitions passes through the interrogation region at a first timerange, generating high and low peaks in signal intensity for the one ormore signal channels corresponding to emission by at least one componentin the partitions of the at least one dispersed phase in the firstsample. The first sample is followed by a spacer fluid that does notcomprise a component that emits electromagnetic radiation withrelatively high intensity when exposed to electromagnetic radiation fromthe excitation sources, and the intensity of the signal measured at thedetection element is very low for a second time period. Finally, asecond sample comprising a plurality of partitions passes through theinterrogation region over a third time range, generating high and lowpeaks in signal intensity for the one or more signal channelscorresponding to emission by at least one component in the partitions ofthe at least one dispersed phase in the second sample. The first timeperiod precedes the second time period which precedes the third timeperiod, and the length of the second period, where intensity is below athreshold value for a major fraction of the second time period,indicates a boundary between samples. In certain embodiments, the majorfraction is at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, at least 99.9%, or at least 99.99% of the timein the second time interval. In certain embodiments, the length of thesecond time interval is at least 1 second, at least 5 seconds, at least10 seconds, at least 30 seconds, at least 60 seconds, or at least 120seconds. FIG. 72B shows another embodiment where the spacer fluidcomprises at least one component that may be excited by at least one ofthe excitation sources. In the second time interval, the signal of atleast one of the detection channels increases to a value above athreshold value for a major fraction of the second time interval. Incertain embodiments, the major fraction is at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%,or at least 99.99% of the time in the second time interval. In certainembodiments, the wavelength range at which the at least one component ofthe spacer fluid is excited coincides with at least one wavelength rangeat which at least one component of the partitions of the at least onedispersed phase are excited. In other embodiments, the wavelength rangeat which the at least one component of the spacer fluid is excited doesnot coincide with at least one wavelength range at which at least onecomponent of the partitions of the at least one dispersed phase isexcited. The arrangement where the excitation ranges do overlap allowsfor the use of one of the detection channels for multiple purposes.

FIG. 73—Fiber excitation on tube. FIG. 73 shows a system for providingradiant energy to partitions. The system comprises a conduit 7301 thatcomprises an inlet 7302 and an outlet 7303 and an interrogation region7304. The system additionally comprises a first excitation source 7305that comprises a focusing element 7306 and an optical fiber 7307. Thefirst excitation source provides radiant energy in a first wavelengthrange to excite one or more components of partitions of at least onedispersed phase flowing in the interrogation region 7304. The focusingelement 7306 focuses radiant energy into an optical fiber 7307 that maybe focused onto the interrogation region 7304. In some embodiments, thesystem additionally comprises a second excitation source 7308 thatcomprises a second focusing element 7309 and a second optical fiber7310, as well as a light combiner 7311 that combines radiant energy intoa fiber optic output 7312 to provide the combined radiant energy fromthe first and second optical sources onto the interrogation region 7304.The system may comprise three, four, or more excitation sources, withall of the radiant energy from the excitation sources provided to theinterrogation region 7304 through the fiber optic output 7312. In someembodiments, the focusing elements comprise lenses. The system mayadditionally comprise an emission collimator 7313, an emission filter7314, an emission focuser 7315, an optical restriction 7316, and adetection element 7317. Energy emitted from partitions in theinterrogation region after excitation by the one or more wavelengthranges provided by the one or more excitation sources is collimated bythe emission collimator 7313 and restricted to a subset of wavelengthsthat preferentially coincide with the wavelengths of relatively largestemission of one or more excited components in the partitions by theemission filter 7314. Radiant energy leaving the emission filter 7314 isfocused on the aperture of the optical restriction 7316, through whichpasses radiant energy substantially originating from a single partition.The radiant energy passing through the optical restriction 7316 isincident on the detection element 7317, where a property of the radiantenergy is detected. In some embodiments, the property is a radiantintensity or a radiant power. In some embodiments, the light combiner7311 additionally comprises an excitation filter to limit the range ofwavelengths passing through the fiber optic output 7312 to those thatsubstantially coincide with wavelengths that will excite one or morecomponents in the partitions passing through the interrogation region.In some embodiments, one or more excitation source may be a laser or alight emitting diode.

FIG. 74—two detector sample boundary detection. FIG. 74 shows a systemfor detecting a sample boundary. The system comprises a conduit 7101, aninlet 7102, an outlet 7103, a first interrogation region 7104, a secondinterrogation region 7105, a first electromagnetic radiation detector7406, and a second electromagnetic radiation detector 7407. At least onecontinuous phase and partitions of at least one dispersed phase enterthe conduit 7101 at the inlet 7102, flow through the first interrogationregion 7104, the second interrogation region 7105, and out the outlet7103. A first electromagnetic radiation detector 7406 detects an opticalproperty of partitions of the at least one dispersed phase thatindicates the boundary between two samples. The second electromagneticradiation detector 7407 measures at least one additional opticalproperty of the partitions of the at least one dispersed phase relatedto a property of at least one component of the partitions of the atleast one dispersed phase. In certain embodiments, the firstelectromagnetic radiation detector 7406 is positioned to measure theabsorbance of electromagnetic radiation through the partitions of the atleast one dispersed phase. In certain of these embodiments, the spacerfluid comprises a component that preferentially absorbs electromagneticradiation in a wavelength range, and a decrease in the electromagneticradiation measured by the detector 7406 indicates a sample boundary. Incertain further embodiments, the wavelength range is infrared. Incertain embodiments, the samples comprise a component thatpreferentially absorbs electromagnetic radiation in a wavelength range,and an increase in the electromagnetic radiation measured by thedetector 7406 indicates a sample boundary. In other embodiments, theelectromagnetic radiation detector 7406 comprises an excitation sourceand an emission detector, and the excitation source excites a componentin the partitions of the at least one dispersed phase to emitelectromagnetic radiation in a wavelength range. In further embodiments,the spacer fluid comprises a component that is excited byelectromagnetic radiation emitted by the excitation source and anincrease in the electromagnetic radiation measured by theelectromagnetic radiation detector 7406 indicates a boundary betweensamples. In other embodiments, the samples comprise at least onecomponent that is excited by the electromagnetic radiation emitted bythe excitation source and a minimum period of time where the intensityor power of the electromagnetic radiation measured by the detector 7406is below a threshold indicates the boundary between samples.

FIGS. 50 and 75 Star-shaped detector. FIG. 50 shows a detectorarrangement that might be used for lock-in amplification of opticalsignals from partitions. As shown, the embodiment comprises up to fourexcitation sources but only a single detection source arranged around acentral tube. This shows a key advantage of lock-in detection, i.e. thata single detection element may measure emission from multiple excitedcomponents in a partition of a dispersed phase. Other embodiments, asdescribed elsewhere herein, may comprise any number of excitationsources and/or detection elements arranged around a tubularinterrogation region. FIG. 75 shows a system for detecting emission ofradiant energy from partitions using one or more detectors. The systemcomprises an interrogation tube 7501, a first excitation source 7502,and a detection element 7503. As described elsewhere herein, the firstexcitation source may comprise collimating elements, filters, and/orfocusing elements to focus radiant energy on a partition in theinterrogation tube 7501 of a first wavelength range A so as to exciteone or more components in the partition to emit energy in a secondwavelength range A′. The detection element 7503 may additionallycomprise an optical restriction, collimating elements, filters, and/orfocusing elements to collect radiant energy in the second wavelengthrange A′ from a single partition in the interrogation tube 7501 andmeasure an optical property of at least one component in the partition.In some embodiments, the first excitation source 7502 is substantiallycoplanar with a plane normal to the axis of flow in the interrogationtube 7501. In some embodiments, lock-in amplification is used to improvea signal-to-noise ratio. In some embodiments, the system may comprise asecond excitation source 7504, a third excitation source 7505, and/or afourth excitation source 7506, or more excitation sources, as needed. Insome embodiments, lock-in amplification may be used to measure emissionof radiant energy excited by at least two excitation sources, asdescribed herein. As described elsewhere herein, the detection element7503 may be any suitable measurement element. In some embodiments, thedetection element 7503 comprises a photomultiplier tube, a siliconphotomultiplier, a photodiode, a avalanche photodiode, a charge coupleddevice, or an array of charge coupled devices.

An optical stage (interrogation region0 may be used for measuringdroplets flowing through systems as described herein. In some instances,droplet flow through a tube substantially parallel to a plane of thepinhole and near a common optical focal point of the two lenses. Aninternal diameter of the tube may allow free-flow spherical diameter ofthe droplets to have a larger internal diameter. In some instances, theoptical stage is a microchannel on a microfluidic chip. In someinstances, a region of the microchannel where a dimension of themicrochannel is smaller than a spherical diameter of the droplets is aregion for the excitation and emission lenses. The optical stage maycomprise a material with a greater affinity for the continuous phasethan the dispersed phase.

V. Definitions

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this system and methods belongs.

As used herein, the term “comprising” and its grammatical equivalentsspecifies the presence of stated features, integers, steps, operations,elements, and/or components, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components, and/or groups thereof. As used herein, the term“and/or” includes any and all combinations of one or more of theassociated listed items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers +/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range. As used herein, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

As used herein, the terms “amplifying” and “amplification” are usedinterchangeably and generally refer to producing one or more copies of anucleic acid.

As used herein, the terms “nucleic acid” and “nucleic acid molecule” areused interchangeably and generally refer to a polymeric form ofnucleotides of any length, either deoxyribonucleotides (dNTPs) orribonucleotides (rNTPs), or analogs thereof. Nucleic acids may have anythree dimensional structure, and may perform any function, known orunknown. Non-limiting examples of nucleic acids include deoxyribonucleicacid (DNA), ribonucleic acid (RNA), a peptide nucleic acid (PNA), codingor non-coding regions of a gene or gene fragment, loci (locus) definedfrom linkage analysis, exons, introns, messenger RNA (mRNA), transferRNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA(shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant nucleic acids,branched nucleic acids, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers. Anucleic acid may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs such as, for example,locked nucleic acids (LNA), fluorinated nucleic acids (FNA), bridgednucleic acids and thio-nucleotides. If present, modifications to thenucleotide structure may be made before or after assembly of the nucleicacid. The sequence of nucleotides of a nucleic acid may be interruptedby non-nucleotide components, such as, for example a linker or othertype of spacer. A nucleic acid may be further modified afterpolymerization, such as by conjugation or binding with a detectablespecies. In some instances, a nucleic acid may be a primer that, in someembodiments, can be used to amplify another nucleic acid molecule.

As used herein, the term “primer” generally refers to a nucleic acidmolecule that is capable of hybridizing with a template nucleic acidmolecule and capable of being extended in a template-directed manner viathe template nucleic acid molecule.

As used herein, the terms “target nucleic acid” and “target nucleic acidmolecule” are used interchangeably and generally refer to a nucleic acidmolecule in a starting population of nucleic acid molecules having atarget sequence whose presence, amount, and/or nucleotide sequence, orchanges in one or more of these, are desired to be determined. In someinstances, a target nucleic acid molecule may be double-stranded. Insome instances, a target nucleic acid molecule may be single-stranded.In general, the term “target nucleic acid strand” refers to asingle-stranded target nucleic acid molecule. In general, the term“target nucleic acid sequence” refers to a nucleic acid sequence on astrand of target nucleic acid. A target nucleic acid molecule or targetnucleic acid sequence can be a portion of a gene, a regulatory sequence,genomic DNA, cDNA, RNA including mRNA, miRNA, rRNA, or others. Thetarget nucleic acid sequence or target nucleic acid molecule can be atarget nucleic acid sequence or target nucleic acid molecule from asample or a secondary target such as a product of an amplificationreaction.

As used herein, the term “sample” includes any volume of a dispersedphase to be processed in the process system. Samples may or may notcomprise a sub-volume of a larger volume. A sample may include chemicalor biological components to be one or more of categorized, sorted,analyzed, assayed for one or more qualitative or quantitativeproperties, reacted, separated, or subjected to physical changes intemperature, pressure, phase, state, electric potential, pH, or anyother properties of interest. Samples may comprise a hydrophilicmaterial (e.g. water) to be dispersed in a continuous phase comprising ahydrophobic material (e.g. oil), or may comprise a hydrophobic material(e.g. an oil) to be dispersed in a continuous phase comprising ahydrophilic material (e.g. water). Samples may additionally comprisebiochemical or chemical reagents, catalysts, co-factors, co-solvents,buffers, salts, enzymes, dyes, reporter systems, or any other componentsthat would aid in the desired process or processes to be conducted onthe samples in the process system.

As used herein the term “partition” includes any subset of a volume of adispersed phase dispersed in a continuous phase. Partitions may be anyvolume up to the volume of the original volume of a dispersed phase. Theterm “droplet,” as used herein, is taken to mean partition.

As used herein the term “dispersed phase” includes any fluid that, whenflowing in a conduit in the process system in contact with a continuousphase, will be nearly completely or substantially completely surroundedby fluid of a continuous phase. As such, dispersed phase refers tofluids even before they are actually dispersed in the continuous phase,as some or all of the volume of fluid referred to as dispersed phasebecomes surrounded by a continuous phase when in the process system.Dispersed phase may include sample and/or spacer fluid, as defined inthe present systems and methods. Dispersed phase may be in one or morevolumes or partitions. Dispersed phase includes fluids nearly completelyor completely immiscible with a continuous phase.

As used herein the term “continuous phase” includes any fluid that, whenflowing in a conduit in the process system, it nearly or substantiallycompletely surrounds fluids of dispersed phases such that the fluids ofthe dispersed phase become entrained in the continuous phase flow. Assuch, continuous phase refers to fluids even before they are actually incontact with dispersed phase fluids, as some or all of the volume offluid referred to as continuous phase nearly completely or completelysurrounds volumes of dispersed phase when in the process system.

VI. Numbered Embodiments

Numbered embodiment 1 comprises a system for producing a serial flowemulsion comprising (i) an intake system to sequentially transport aplurality of separate samples or portions of samples from a series ofsample containers; (ii) a process system, wherein the process systemcomprises a partitioner to generate a plurality of partitions in acontinuous phase from each of the samples; and (iii) an injectorpositioned between the intake system and the process system, wherein theinjector is configured to be in fluid communication with the intakesystem, or to be in fluid communication with the process system, but notboth simultaneously. Numbered embodiment 2 comprises the system ofnumbered embodiment 1 and further comprises (iii) a reactor fluidlyconnected to the partitioner to initiate or modulate one or morereactions in one or more of the partitions. Numbered embodiment 3comprises the system of numbered embodiments 1-2 wherein the reactor isconfigured so all partitions flow through the reactor. Numberedembodiment 4 comprises the system of numbered embodiments 1-3 whereinthe reactor supplies electromagnetic radiation, heating, cooling, sonicenergy, or particulate radiation or a combination thereof to thepartitions. Numbered embodiment 5 comprises the system of numberedembodiments 1-4 wherein the reactor comprises at least one thermal zoneat a set temperature. Numbered embodiment 6 comprises the system ofnumbered embodiments 1-5 wherein the reactor comprises at least twothermal zones wherein each thermal zone is at a different temperature.Numbered embodiment 7 comprises the system of numbered embodiments 1-6wherein the reactor comprises a conduit fluidly connected to thepartitioner through which partitions flow, wherein the conduitrepeatedly contacts the at least two different thermal zones. Numberedembodiment 8 comprises the system of numbered embodiments 1-7 whereinthe conduit is arranged as a helix around a central core, wherein thecentral core comprises at least two different thermal zones at twodifferent temperatures, and wherein the conduit is wrapped around thecore so as to contact the core at the at least two different thermalzones. Numbered embodiment 9 comprises the system of numberedembodiments 1-8 further comprising (iv) a detector fluidly connected tothe reactor to detect a characteristic of one or more of the partitions.Numbered embodiment 10 comprises the system of numbered embodiments 1-9wherein the intake system is configured to be cleaned between sampleswithout contact with the process system. Numbered embodiment 11comprises the system of numbered embodiments 1-10 wherein the intakesystem is configured to be fluidly connected to at least one of (a) apurge fluid reservoir; (b) a denaturing fluid reservoir; and/or (c) aspacer fluid reservoir. Numbered embodiment 12 comprises the system ofnumbered embodiments 1-11 wherein the detector comprises at least one of(a) an optical restriction that limits the amount of electromagneticradiation that reaches a detection element in the detector from aninterrogation region in the detector where partitions are detected toless than 10% of the electromagnetic radiation that would reach thedetection element without the optical restriction; (b) an interrogationregion comprising a conduit wherein partitions flow in single filethrough the conduit, and wherein the cross-sectional area of the conduitis less than 90% of the average spherical cross-sectional area of thepartitions; (c) an excitation source to provide electromagneticradiation to the interrogation region and a system to provide lock-inamplification of the excitation source; (d) a system to separatepartitions prior to the interrogation region of the detector. Numberedembodiment 13 comprises the system of numbered embodiments 1-12 whereinthe detector comprises at least two of (a) an optical restriction thatlimits the amount of electromagnetic radiation that reaches a detectionelement in the detector from an interrogation region in the detectorwhere partitions are detected to less than 10% of the electromagneticradiation that would reach the detection element without the opticalrestriction; (b) an interrogation region comprising a conduit whereinpartitions flow in single file through the conduit, and wherein thecross-sectional area of the conduit is less than 90% of the averagespherical cross-sectional area of the partitions; (c) an excitationsource to provide electromagnetic radiation to the interrogation regionand a system to provide lock-in amplification of the excitation source;(d) a system to separate partitions prior to the interrogation region ofthe detector. Numbered embodiment 14 comprises the system of numberedembodiments 1-13 wherein the detector comprises at least three of (a) anoptical restriction that limits the amount of electromagnetic radiationthat reaches a detection element in the detector from an interrogationregion in the detector where partitions are detected to less than 10% ofthe electromagnetic radiation that would reach the detection elementwithout the optical restriction; (b) an interrogation region comprisinga conduit wherein partitions flow in single file through the conduit,and wherein the cross-sectional area of the conduit is less than 90% ofthe average spherical cross-sectional area of the partitions; (c) anexcitation source to provide electromagnetic radiation to theinterrogation region and a system to provide lock-in amplification ofthe excitation source; (d) a system to separate partitions prior to theinterrogation region of the detector. Numbered embodiment 15 comprisesthe system of numbered embodiments 1-14 wherein the intake systemcomprises a sampler, wherein the sampler is configured to remove asample or a portion of a sample from a sample container and transportthe sample or portion of sample to the injector, and the injector isconfigured to inject a fixed volume of the sample or portion of sampleinto a conduit fluidly connected to the process system. Numberedembodiment 16 comprises the system of numbered embodiments 1-15 whereinthe intake system is configured to provide a sample comprising a firstfluid to the process system and the process system is configured toprovide a second fluid to the system after the first fluid, and whereinat least 80, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 99.99% surfaces of allcomponents of the system that come into contact with the sample orportions of the sample have greater affinity for the second fluid thanfor the first fluid. Numbered embodiment 17 comprises a methodcomprising (i) transporting a first sample comprising a first dispersedphase from a first sample container into an intake system; (ii) flowingthe first sample from the intake system to an injector that is fluidlyconnected to the injection system but not to a process system; (iii)repositioning the injector so that it is fluidly connected to theprocess system but not the intake system; (iv) flowing the first samplefrom the injector into the process system; (v) partitioning the firstsample into a plurality of partitions of the first dispersed phase in acontinuous phase; (vi) repositioning the injector so that it is fluidlyconnected to the injection system but not to the process system; (vii)transporting a second sample comprising a second dispersed phase from afirst second container into the intake system and into the injector;(viii) repositioning the injector so that it is fluidly connected to theprocess system but not the intake system; (ix) flowing the second samplefrom the injector into the process system; and (x) partitioning thesecond sample into a plurality of partitions of the second dispersedphase in the continuous phase. Numbered embodiment 18 comprises themethod of numbered embodiment 17 comprising cleaning the intake systemcomprises at least one of (a) purging remaining sample from the intakesystem; and/or (b) denaturing remaining sample in the intake system.Numbered embodiment 19 comprises the method of numbered embodiment 17-18wherein cleaning the intake system comprises both of (a) purgingremaining sample from the intake system; and (b) denaturing remainingsample in the intake system. Numbered embodiment 20 comprises the methodof numbered embodiments 17-19 further comprising transporting a spacerfluid from a spacer fluid reservoir into the intake system, and flowingthe spacer fluid into the process system, wherein the spacer fluid isflowed into the process system between the first and second samples.Numbered embodiment 21 comprises the method of numbered embodiments17-20 further comprising flowing the partitions of the first and secondsamples in a reactor for initiating and/or modulating one or morereactions in one or more of the partitions. Numbered embodiment 22comprises the method of numbered embodiments 17-21 further comprisingflowing the partitions through a detector fluidly connected to thereactor to detect one or more of the partitions. Numbered embodiment 23comprises the method of numbered embodiments 17-22 wherein the detectorcomprises at least one of (a) an optical restriction that limits theamount of electromagnetic radiation that reaches a detection element inthe detector from an interrogation region in the detector wherepartitions are detected to less than 10% of the electromagneticradiation that would reach the detection element without the opticalrestriction; (b) an interrogation region comprising a conduit whereinpartitions flow in single file through the conduit, and wherein thecross-sectional area of the conduit is less than 90% of the averagespherical cross-sectional area of the partitions; (c) an excitationsource to provide electromagnetic radiation to the interrogation regionand a system to provide lock-in amplification of the excitation source;(d) a system to separate partitions prior to the interrogation region ofthe detector. Numbered embodiment 24 comprises the method of numberedembodiments 17-23 wherein the detector comprises at least two of (a) anoptical restriction that limits the amount of electromagnetic radiationthat reaches a detection element in the detector from an interrogationregion in the detector where partitions are detected to less than 10% ofthe electromagnetic radiation that would reach the detection elementwithout the optical restriction; (b) an interrogation region comprisinga conduit wherein partitions flow in single file through the conduit,and wherein the cross-sectional area of the conduit is less than 90% ofthe average spherical cross-sectional area of the partitions; (c) anexcitation source to provide electromagnetic radiation to theinterrogation region and a system to provide lock-in amplification ofthe excitation source; (d) a system to separate partitions prior to theinterrogation region of the detector. Numbered embodiment 25 comprisesthe method of numbered embodiments 17-24 wherein the detector comprisesat least three of (a) an optical restriction that limits the amount ofelectromagnetic radiation that reaches a detection element in thedetector from an interrogation region in the detector where partitionsare detected to less than 10% of the electromagnetic radiation thatwould reach the detection element without the optical restriction; (b)an interrogation region comprising a conduit wherein partitions flow insingle file through the conduit, and wherein the cross-sectional area ofthe conduit is less than 90% of the average spherical cross-sectionalarea of the partitions; (c) an excitation source to provideelectromagnetic radiation to the interrogation region and a system toprovide lock-in amplification of the excitation source; (d) a system toseparate partitions prior to the interrogation region of the detector.Numbered embodiment 26 comprises the method of numbered embodiments17-25 further comprising performing a reaction in at least a portion ofthe partitions. Numbered embodiment 27 comprises the method of numberedembodiments 17-26 the portion of the partitions comprise at least onenucleic acid per partition, and the reaction is a polymerase chainreaction (PCR). Numbered embodiment 28 comprises the method of numberedembodiments 17-27 wherein the samples comprise a first fluid and theprocess system provides a second fluid to the system after the firstfluid, and wherein at least 80, 90, 95, 96, 97, 98, 99, 99.5, 99.9,99.99% surfaces of all components of the system that come into contactwith the sample or portions of the sample have greater affinity for thesecond fluid than for the first fluid. Numbered embodiment 29 comprisesa system for conducting a serial flow emulsion reaction comprisingcomponents, the system comprising (i) an intake system to sequentiallytransport a plurality of separate samples or portions of samples from aseries of sample containers; (ii) a process system, wherein the processsystem comprises a reactor to initiate or modulate a reaction in thesamples or portions of the samples; and (iii) an injector positionedbetween the intake system and the process system, wherein the injectoris configured to be in fluid communication with the intake system, or tobe in fluid communication with the process system, but not bothsimultaneously. Numbered embodiment 30 comprises the system of numberedembodiment 29 wherein the reactor supplies electromagnetic radiation,heating, cooling, sonic energy, particulate radiation or a combinationthereof to the one or more portions of the sample. Numbered embodiment31 comprises the system of numbered embodiments 29-30 further comprising(iv) a detector fluidly connected to the reactor to detect acharacteristic of the one or more portions of the sample. Numberedembodiment 32 comprises the system of numbered embodiments 29-31 whereinthe detector comprises at least one of Numbered embodiment 23 comprisesthe method of numbered embodiments 17-22 wherein the detector comprisesat least one of (a) an optical restriction that limits the amount ofelectromagnetic radiation that reaches a detection element in thedetector from an interrogation region in the detector where partitionsare detected to less than 10% of the electromagnetic radiation thatwould reach the detection element without the optical restriction; (b)an interrogation region comprising a conduit wherein partitions flow insingle file through the conduit, and wherein the cross-sectional area ofthe conduit is less than 90% of the average spherical cross-sectionalarea of the partitions; (c) an excitation source to provideelectromagnetic radiation to the interrogation region and a system toprovide lock-in amplification of the excitation source; (d) a system toseparate partitions prior to the interrogation region of the detector.Numbered embodiment 33 comprises the system of numbered embodiments29-32 wherein the detector comprises at least two of Numbered embodiment23 comprises the method of numbered embodiments 17-22 wherein thedetector comprises at least one of (a) an optical restriction thatlimits the amount of electromagnetic radiation that reaches a detectionelement in the detector from an interrogation region in the detectorwhere partitions are detected to less than 10% of the electromagneticradiation that would reach the detection element without the opticalrestriction; (b) an interrogation region comprising a conduit whereinpartitions flow in single file through the conduit, and wherein thecross-sectional area of the conduit is less than 90% of the averagespherical cross-sectional area of the partitions; (c) an excitationsource to provide electromagnetic radiation to the interrogation regionand a system to provide lock-in amplification of the excitation source;(d) a system to separate partitions prior to the interrogation region ofthe detector. Numbered embodiment 34 comprises the system of numberedembodiments 29-33 wherein the detector comprises at least three ofNumbered embodiment 23 comprises the method of numbered embodiments17-22 wherein the detector comprises at least one of (a) an opticalrestriction that limits the amount of electromagnetic radiation thatreaches a detection element in the detector from an interrogation regionin the detector where partitions are detected to less than 10% of theelectromagnetic radiation that would reach the detection element withoutthe optical restriction; (b) an interrogation region comprising aconduit wherein partitions flow in single file through the conduit, andwherein the cross-sectional area of the conduit is less than 90% of theaverage spherical cross-sectional area of the partitions; (c) anexcitation source to provide electromagnetic radiation to theinterrogation region and a system to provide lock-in amplification ofthe excitation source; (d) a system to separate partitions prior to theinterrogation region of the detector. Numbered embodiment 35 comprisesthe system of numbered embodiments 29-34 wherein the intake system isconfigured to be cleaned between samples while the injector isconfigured to be in fluid communication with the intake system. Numberedembodiment 36 comprises the system of numbered embodiments 29-35 whereinthe intake system is configured to be transiently fluidly connected toat least one of (a) a purge fluid reservoir; (b) a denaturing fluidreservoir; and/or (c) a spacer fluid reservoir. Numbered embodiment 37comprises the system of numbered embodiments 29-36 wherein the injectoris configured to inject a fixed volume. Numbered embodiment 38 comprisesthe system of numbered embodiments 29-37 wherein the injector isconfigured to be fluidly connected to the intake system to receive asample or a portion of a sample, or fluidly connected to the processsystem to move sample or a portion of sample into the process system,but not both. Numbered embodiment 39 comprises the system of numberedembodiments 29-38 further comprising a partitioner fluidly connected onan upstream side to the intake system and on a downstream side to thereactor, for portioning the sample or portion of sample into a pluralityof partitions to be flowed through the reactor. Numbered embodiment 40comprises the system of numbered embodiments 29-39 wherein the intakesystem is configured to provide a sample comprising a first fluid to theprocess system and the process system is configured to provide a secondfluid to the system after the first fluid, and wherein at least 80, 90,95, 96, 97, 98, 99, 99.5, 99.9, 99.99% surfaces of all components of thesystem that come into contact with the sample or portions of the samplehave greater affinity for the second fluid than for the first fluid.Numbered embodiment 41 comprises a system comprising (i) an intakesystem for removing a series of samples from a series of samplecontainers, fluidly connected to (ii) an injector for injecting at leasta portion of the sample from the sampler into a process system, whereinone, two, three, four, five, six, or seven of the series of samplecontainers comprises a barrier for each sample container to separate itfrom the environment, the intake system is configured to clean anexternal surface of an intake conduit of the intake system that comes incontact with sample, at least 90% of surfaces of the system that come incontact with sample comprising a first fluid have a lower affinity forthe first fluid than for a second fluid with which the surfaces come incontact, the intake system is configured to be cleaned between samples,the system is configured to add a spacing fluid between samples in theseries of samples, conduits in the system are configured to allowlaminar flow, or a conduit in the system between a partitioner and apartition separation system or a partitioner and a detector are orientedso that flow in the conduit is within 20 degrees of orthogonal togravity, or a combination thereof. Numbered embodiment 42 comprises thesystem of numbered embodiment 41 wherein the injector is configured tobe isolated from the process system during a cleaning phase. Numberedembodiment 43 comprises the system of numbered embodiments 41-42 furthercomprising a reservoir comprising purge fluid operably connected to theintake system, the injector, or both the intake system and the injector.Numbered embodiment 44 comprises a system comprising a detector fordetecting one or more partitions in a flowing series of partitionscomprising (i) a conduit configured to flow the partitions within theconduit in single file, comprising an interrogation region whereinpartitions are detected; (ii) a detection element for detecting theelectromagnetic radiation emitted by the partitions in the interrogationregion, if present; and (iii) an optical restriction configured andpositioned between the interrogation region and the detection element sothat only a portion of the electromagnetic radiation from theinterrogation region that can be detected by the detection element isactually detected. Numbered embodiment 45 comprises the system ofnumbered embodiment 44 wherein the portion of electromagnetic radiationthat can be detected due to the optical restriction is less than 10% ofthe amount that would be detected without the optical restriction.Numbered embodiment 46 comprises the system of numbered embodiments44-45 wherein the interrogation region has a cross-sectional area equalto or less than 90% of the average spherical cross-sectional area of thepartitions. Numbered embodiment 47 comprises the system of numberedembodiments 44-46 wherein the interrogation region has a cross-sectionalarea equal to or less than 50% of the average spherical cross-sectionalarea of the partitions. Numbered embodiment 48 comprises the system ofnumbered embodiments 44-47 further comprising (iv) an excitation sourcefor supplying electromagnetic radiation to the interrogation region ofthe conduit. Numbered embodiment 49 comprises the system of numberedembodiments 44-48 comprising a plurality of excitation sources, each ofwhich supplies electromagnetic radiation to the interrogation region.Numbered embodiment 50 comprises the system of numbered embodiments44-49 wherein the excitation source or sources comprises a lock-inamplifier. Numbered embodiment 51 comprises the system of numberedembodiments 44-50 further comprising a partitioner fluidly connected tothe detector, wherein the partitioner is configured to generatepartitions of dispersed phase in a continuous phase from a sample.Numbered embodiment 52 comprises the system of numbered embodiments44-51 further comprising a reactor for initiating or modulating areaction in the partitions. Numbered embodiment 53 comprises the systemof numbered embodiments 44-52 wherein at least a portion of thepartitions comprise a single nucleic acid molecule and the reactorcomprises a thermal cycler for polymerase chain reaction (PCR)reactions. Numbered embodiment 54 comprises the system of numberedembodiments 44-53 wherein the partitioner is configured to producepartitions of an average volume of 0.05-50 nL. Numbered embodiment 55comprises the system of numbered embodiments 44-54 further comprising apartition separation system configured to add a separation fluid to theflow of partitions prior to the interrogation region of the detector.Numbered embodiment 56 comprises the system of numbered embodiments44-55 further comprising an intake system that can be fluidly connectedto a sample container and an injector that can be fluidly connected tothe intake system or fluidly connected to the partitioner for supplyingsample to the partitioner. Numbered embodiment 57 comprises the systemof numbered embodiments 44-56 wherein the injector is configured toinject a fixed volume. Numbered embodiment 58 comprises a method fordetecting partitions continuously flowing through an interrogationregion of a conduit comprising detecting at least one detectablecomponent in single partitions as they flow through the interrogationregion by detecting electromagnetic radiation emitted from thedetectable component by a detection element, wherein an opticalrestriction is configured and positioned between the interrogationregion and the detection element so that only a portion of theelectromagnetic radiation from the interrogation region that can bedetected by the detection element is actually detected. Numberedembodiment 59 comprises the method of numbered embodiment 58 wherein theportion of electromagnetic radiation that can be detected due to theoptical restriction is less than 10% of the amount that would bedetected without the optical restriction. Numbered embodiment 60comprises the method of numbered embodiments 58-59 wherein theinterrogation region of the conduit is configured to have across-sectional area equal to or less than 100, 95, 90, 80, 70, 60, 50,40, 30 20, 10%, for example less than 90%, such as less than 50% theaverage cross-sectional area of the partitions. Numbered embodiment 61comprises the method of numbered embodiments 58-60 further comprisingsupplying electromagnetic radiation from an excitation source to theinterrogation region. Numbered embodiment 62 comprises the method ofnumbered embodiments 58-61 further comprising performing lock-inamplification on the electromagnetic radiation from the excitationsource. Numbered embodiment 63 comprises the method of numberedembodiments 58-62 comprising supplying electromagnetic radiation from aplurality of excitation sources to the interrogation region, andperforming lock-in amplification on the plurality of excitation sources.Numbered embodiment 64 comprises the method of numbered embodiments58-63 further comprising partitioning a sample into the partitions.Numbered embodiment 65 comprises the method of numbered embodiments58-64 wherein the partitioning produces partitions of an average volumeof 0.05-50 nL. Numbered embodiment 66 comprises the method of numberedembodiments 58-65 further comprising initiating or modulating one ormore reactions in the partitions in a reactor. Numbered embodiment 67comprises the method of numbered embodiments 58-66 wherein at least aportion of the partitions comprise a single nucleic acid molecule andthe reactor provides thermal cycling to perform PCR on the nucleic acid.Numbered embodiment 68 comprises the method of numbered embodiments58-67 further comprising adding a separation fluid to the flow ofpartitions before they reach the interrogation region. Numberedembodiment 69 comprise a system comprising a detector for detecting oneor more partitions in a flowing series of partitions comprising (i) aconduit configured to flow the partitions within the conduit in singlefile, comprising an interrogation region; (ii) one or more excitationsources for supplying electromagnetic radiation to the interrogationregion, wherein, if a plurality of excitation sources is used, each ofthe excitation sources supplies electromagnetic radiation at a differentwavelength, wherein the different wavelengths excite one or moredifferent molecules in the partition, if present; (iii) a detectionelement for detecting electromagnetic radiation emitted by themolecules, if present, in response to the electromagnetic radiation fromthe one or more excitation sources; wherein the excitation source orsources are configured to provide lock-in amplification. Numberedembodiment 70 comprises the system of numbered embodiment 69 comprisingat least 2, 3, 4, 5, or 6, such as at least 3, different excitationsources, supplying electromagnetic radiation at 2, 3, 4, 5, or 6, suchas at least 3 different wavelengths. Numbered embodiment 71 comprisesthe system of numbered embodiments 69-70 wherein the detection elementcomprises an avalanche photodiode. Numbered embodiment 72 comprises thesystem of numbered embodiments 69-71 further comprising (iv) an opticalrestriction configured and positioned between the interrogation regionand the detection element so that only a portion of the electromagneticradiation, such as less than 10% of the electromagnetic radiation, fromthe interrogation region that can be detected by the detection elementis actually detected. Numbered embodiment 73 comprises the system ofnumbered embodiments 69-72 wherein the interrogation region has across-sectional area equal to or less than 90%, 80, 70, 60, 50, 40, 30,20, or 10%, such as less than 90%, for example, less than 50%, of theaverage spherical cross-sectional area of the partitions. Numberedembodiment 74 comprises the system of numbered embodiments 69-73 furthercomprising a partitioner fluidly connected to the detector, wherein thepartitioner is configured to generate partitions of dispersed phase in acontinuous phase from a sample. Numbered embodiment 75 comprises thesystem of numbered embodiments 69-74 further comprising a reactor forinitiating or modulating a reaction in the partitions. Numberedembodiment 76 comprises the system of numbered embodiments 69-75 whereinat least a portion of the partitions comprise a single nucleic acidmolecule and the reactor comprises a thermal cycler for polymerase chainreaction (PCR) reactions. Numbered embodiment 77 comprises the system ofnumbered embodiments 69-76 wherein the partitioner is configured toproduce partitions of an average volume of 0.05-50 nL. Numberedembodiment 78 comprises the system of numbered embodiments 69-77 furthercomprising a partition separation system configured to add a separationfluid to the flow of partitions prior to the interrogation region of thedetector. Numbered embodiment 79 comprises the system of numberedembodiments 69-78 further comprising an intake system fluidly connectedto at least one sample container and to an injector, wherein theinjector is configured to reposition to move at least part of sample tothe partitioner. Numbered embodiment 80 comprises the system of numberedembodiments 69-79 wherein the injector is configured to inject a fixedvolume. Numbered embodiment 81 comprises a method for detectingmolecules in partitions flowing through a conduit comprising (i) flowingthe partitions through an interrogation region in the conduit in singlefile; (ii) supplying electromagnetic radiation to the interrogationregion from one or more excitation sources wherein, if a plurality ofexcitation sources is used, each of the excitation sources supplieselectromagnetic radiation at a different wavelength, wherein thedifferent wavelengths excite one or more different molecules in thepartition, if present; and (iii) detecting electromagnetic radiationemitted by the molecules, if present, in response to the electromagneticradiation from one or more of the excitation sources with a detectionelement, wherein the excitation source or sources provide lock-inamplification. Numbered embodiment 82 comprises the method of numberedembodiment 81 comprising supplying electromagnetic radiation from atleast 2, 3, 4, 5, 6, such as at least 3, different excitation sources,supplying electromagnetic radiation at 2, 3, 4, 5, 6, such as at least 3different wavelengths. Numbered embodiment 83 comprises the method ofnumbered embodiments 81-82 wherein an optical restriction is configuredand positioned between the interrogation region and the detectionelement so that only a portion, for example less than 10%, such as lessthan 5%, of the electromagnetic radiation from the interrogation regionthat can be detected by the detection element is actually detected.Numbered embodiment 84 comprises the method of numbered embodiments81-83 wherein the interrogation region has a cross-sectional area equalto or less than 90%, 80, 70, 60, 50, 40, 30, 20, or 10%, such as lessthan 90%, for example, less than 50%, of the average sphericalcross-sectional area of the partitions. Numbered embodiment 85 comprisesthe method of numbered embodiments 81-84 further comprising partitioninga sample into the partitions of dispersed phase in a continuous phase.Numbered embodiment 86 comprises the method of numbered embodiments81-85 wherein the average volume of the partitions is 0.05-50 nL.Numbered embodiment 87 comprises the method of numbered embodiments81-86 comprising initiation or modulating one or more reactions in thepartitions in a reactor. Numbered embodiment 88 comprises the method ofnumbered embodiments 81-87 wherein at least a portion of the partitionscomprise a single nucleic acid molecule and the reactor thermal cyclesthe partitions to perform polymerase chain reaction (PCR) reactions.Numbered embodiment 89 comprises the method of numbered embodiments81-88 further comprising adding a separation fluid to the flow ofpartitions prior to the interrogation region of the detector. Numberedembodiment 90 comprises the method of numbered embodiments 81-89 furthercomprising supplying sample to the partitioner from an intake system viaan injector which can be fluidly coupled to a sample containercontaining the sample, or fluidly coupled to the partitioner, but notboth simultaneously. Numbered embodiment 91 comprises the method ofnumbered embodiments 81-90 further comprising cleaning the intakesystem. Numbered embodiment 92 comprises a system comprising a detectorfor detecting one or more partitions in a flowing series of partitionscomprising (i) a conduit configured to flow the partitions within theconduit in single file, comprising an interrogation region, wherein theinterrogation region is configured to have a cross-sectional area equalto or less than 100, 95, 90, 80, 70, 60, 50, 40, 30 20, 10%, such asequal to or less than 90%, for example equal to or less than 50%, theaverage cross-sectional area of the partitions; (ii) a detection elementfor detecting electromagnetic radiation emitted by the partitions, ifpresent, in the interrogation region. Numbered embodiment 93 comprisesthe system of numbered embodiment 92 further comprising (iii) an opticalrestriction configured and positioned between the interrogation regionand the detection element so that only a portion of the electromagneticradiation, such as less than 10% of the electromagnetic radiation, fromthe interrogation region that can be detected by the detection elementis actually detected. Numbered embodiment 94 comprises the system ofnumbered embodiments 92-93 further comprising (iv) an excitation sourcefor supplying electromagnetic radiation to the interrogation region ofthe conduit. Numbered embodiment 95 comprises the system of numberedembodiments 92-94 comprising a plurality of excitation sources, each ofwhich supplies electromagnetic radiation to the interrogation region.Numbered embodiment 96 comprises the system of numbered embodiments92-95 wherein the excitation source or sources comprises a lock-inamplifier. Numbered embodiment 97 comprises the system of numberedembodiments 92-96 further comprising a partitioner fluidly connected tothe detector, wherein the partitioner is configured to generatepartitions of dispersed phase in a continuous phase from a sample.Numbered embodiment 98 comprises the system of numbered embodiments92-97 further comprising a reactor for initiating or modulating areaction in the partitions. Numbered embodiment 99 comprises the systemof numbered embodiments 92-98 wherein at least a portion of thepartitions comprise a single nucleic acid molecule and the reactorcomprises a thermal cycler for polymerase chain reaction (PCR)reactions. Numbered embodiment 100 comprises the system of numberedembodiments 92-99 wherein the partitioner is configured to producepartitions of an average volume of 0.05-50 nL. Numbered embodiment 101comprises the system of numbered embodiments 92-100 further comprising apartition separation system configured to add a separation fluid to theflow of partitions prior to the interrogation region of the detector.Numbered embodiment 102 comprises the system of numbered embodiments92-101 further comprising an intake system fluidly connected to a samplecontainer and to an injector which can be repositioned to connect to thepartitioner for supplying sample to the partitioner. Numbered embodiment103 comprises the system of numbered embodiments 92-102 wherein theintake system is configured to be cleaned between samples. Numberedembodiment 104 comprises a system comprising a detector for detectingone or more partitions in a flowing series of partitions comprising (i)a conduit configured to flow the partitions within the conduit in singlefile, comprising an interrogation region; (ii) a plurality of excitationsources for supplying electromagnetic radiation to excite moleculeswithin a partition in the conduit, if present, wherein each of theplurality of excitation sources supplies electromagnetic radiation at adifferent wavelength; (iii) a detection element for detectingelectromagnetic radiation emitted by the molecules, if present, inresponse to the electromagnetic radiation from one of the excitationsources; wherein the excitation sources and the emission detector aresituated less than 40, 30, 20, 15, 10, 5, 2, or 1°, such as less than 5degrees, for example less than 2 degrees, away from a plane that isperpendicular to the direction of flow of the partitions through theinterrogation region. Numbered embodiment 105 comprises the system ofnumbered embodiment 104 wherein the conduit is a tube. Numberedembodiment 106 comprises the system of numbered embodiments 104-105wherein the interrogation region is configured to have a cross-sectionalarea equal to or less than 100, 95, 90, 80, 70, 60, 50, 40, 30 20, 10%,such as equal to or less than 90%, such as less than 10%, of the averagecross-sectional area of the partitions. Numbered embodiment 107comprises the system of numbered embodiments 104-106 wherein theplurality of excitation sources comprises at least 2, 3, 4, 5, 6, forexample, at least 3 different excitation sources. Numbered embodiment108 comprises the system of numbered embodiments 104-107 wherein theexcitation sources comprise a lock-in amplifier. Numbered embodiment 109comprises the system of numbered embodiments 104-108 comprising aplurality of emission detectors wherein the excitation sources and theemission detectors are situated less than 40, 30, 20, 15, 10, 5° awayfrom a plane that is perpendicular to the direction of flow of thepartitions through the conduit. Numbered embodiment 110 comprises thesystem of numbered embodiments 104-109 wherein the detection elementcomprises an avalanche photodiode. Numbered embodiment 111 comprises thesystem of numbered embodiments 104-110 further comprising (iv) anoptical restriction configured and positioned between the interrogationregion and the detection element so that only a portion of theelectromagnetic radiation, such as less than 10%, for example less than1% of the electromagnetic radiation, from the interrogation region thatcan be detected by the detection element is actually detected. Numberedembodiment 112 comprises the system of numbered embodiments 104-111further comprising a partitioner fluidly connected to the detector,wherein the partitioner is configured to generate partitions ofdispersed phase in a continuous phase from a sample. Numbered embodiment113 comprises the system of numbered embodiments 104-112 furthercomprising a reactor for initiating or modulating a reaction in thepartitions. Numbered embodiment 114 comprises the system of numberedembodiments 104-113 wherein at least a portion of the partitionscomprise a single nucleic acid molecule and the reactor comprises athermal cycler for polymerase chain reaction (PCR) reactions. Numberedembodiment 115 comprises the system of numbered embodiments 104-114wherein the partitioner is configured to produce partitions of anaverage volume of 0.05-50 nL. Numbered embodiment 116 comprises thesystem of numbered embodiments 104-115 further comprising a partitionseparation system configured to add a separation fluid to the flow ofpartitions prior to the interrogation region of the detector. Numberedembodiment 117 comprises the system of numbered embodiments 104-116further comprising an intake system fluidly connected to a samplecontainer and to an injector, wherein the injector can be repositionedto be fluidly connected to the partitioner, for supplying sample to thepartitioner. Numbered embodiment 118 comprises the system of numberedembodiments 104-117 wherein the injector is configured to inject a fixedvolume. Numbered embodiment 119 comprises a system for producing aserial flow emulsion comprising partitions of dispersed phase in acontinuous phase comprising (i) a partitioner to generate partitionsfrom a sample, wherein the partitioner comprises (a) an first inletoperably connected to a source of the sample, wherein the samplecomprises a first dispersed phase comprising a first fluid, and a firstforce generator operably connected to the sample source to cause thesample to flow through the first inlet; (b) a second inlet operablyconnected to a source of continuous phase, and a second force generatoroperably connected to the continuous phase source to cause thecontinuous phase fluid to flow through the second inlet; and (c) anoutlet; wherein the first and second inlets are positioned to intersectat an angle of no more than 30, 20, 15, 10, 5, 4, 3, 2, 1 degree from180°, for example, an angle of no more than 5 degrees from 180 degreessuch as an angle no more than 1 degree from 180 degrees and the outletis positioned at the intersection of the two inlets and at an anglewithin 20, 15, 10, 5, 4, 3, 2, 1° of 90° from the inlets, for example anangle within 20 degrees of 90 degrees, for example, an angle within 10degrees of 90 degrees. Numbered embodiment 120 comprises the system ofnumbered embodiment 119 further comprising (ii) an intake system toprovide the sample from a sample source; fluidly connected to aninjector, wherein the injector is configured to be fluidly connected tothe intake system, or fluidly connected to the partitioner, but not bothsimultaneously. Numbered embodiment 121 comprises the system of numberedembodiments 119-120 wherein surfaces of the partitioner that come incontact with the sample have a higher affinity for continuous phase thanfor the first fluid. Numbered embodiment 122 comprises an apparatuscomprising a partitioner for portioning a sample into a plurality ofpartitions comprising (i) first inlet channel that carries dispersedphase comprising the sample; (ii) a second inlet channel that carries aflow of a continuous phase, and (iii) a third outlet channel, wherein(a) the axes of first and second inlet channels are oriented within 30°,25°, 20°, 18°, 15°, 13°, 10°, 8°, 5°, 2°, or 1°, such as within 30degrees, of orthogonal to an ambient gravitational field, (b) the firstand second inlet channels intersect at the third outlet channel, and (c)the axis of third outlet channel is oriented within 30°, 25°, 20°, 18°,15°, 13°, 10°, 8°, 5°, 2°, 1°, such as within 30 degrees, of parallelwith the ambient gravitational field, and the direction of flow in thethird channel is opposed to the gravitational field; wherein surfaces ofthe first inlet channel and the outlet channel have greater affinity forthe continuous phase than for the dispersed phase. Numbered embodiment123 comprises the apparatus of numbered embodiment 122 furthercomprising (iv) an intake system to provide the sample from a samplesource; fluidly connected to an injector, wherein the injector isconfigured to be fluidly connected to the intake system, or fluidlyconnected to the partitioner, but not both simultaneously. Numberedembodiment 124 comprises the apparatus of numbered embodiments 122-123wherein surfaces of the partitioner that come in contact with the samplehave a higher affinity for continuous phase than for the first fluid.Numbered embodiment 125 comprises a system for amplifying nucleic acidscomprising (i) an intake system for sampling a sample comprising nucleicacids operably connected to a first inlet channel in a partitioner; (ii)the partitioner, wherein the partitioner comprises the first inletchannel, a second inlet channel that carries a flow of a continuousphase, and a third outlet channel, wherein (a) the axes of first andsecond inlet channels are oriented within 30°, 25°, 20°, 18°, 15°, 13°,10°, 8°, 5°, 2°, or 1 degree, for example, within 30 degrees, oforthogonal to an ambient gravitational field, (b) the first and secondinlet channels intersect at the third outlet channel, and (c) the axisof third outlet channel is oriented within 30°, 25°, 20°, 18°, 15°, 13°,10°, 8°, 5°, 2°, or 1 degree, for example, within 30 degrees, ofparallel with the ambient gravitational field, and the direction of flowin the third channel is opposed to the gravitational field; and (iii) athermal cycler operably connected to the partitioner for thermallycycling the nucleic acids. Numbered embodiment 126 comprises the systemof numbered embodiment 125 wherein the first and second inlet channelsintersect at an angle between 150° and 180°. Numbered embodiment 127comprises a system for conducting a serial flow emulsion processcomprising components comprising (i) an intake system for supplying asample to a process system, wherein the process system comprises apartitioner; (ii) a partitioner to divide the sample into partitions;(iii) a reactor; and (iv) a detector; wherein the intake system can befluidly connected to an injector, wherein the injector is configured tobe fluidly connected to the intake system, or fluidly connected to thepartitioner, but not both simultaneously, and wherein the intake systemis configured to provide a sample comprising a first fluid to the systemand the system is configured to provide a second fluid to the systemafter the first fluid, and wherein at least 80, 90, 95, 99, 99.5, 99.9,99.99%, for example at least 90%, such as at least 99%, of surfaces ofall components of the system that come into contact with the sample havegreater affinity for the second fluid than for the first fluid. Numberedembodiment 128 comprises the system of numbered embodiment 127 whereinthe first fluid and the second fluid are immiscible. Numbered embodiment129 comprises the system of numbered embodiments 127-128 wherein thepartitioner is configured to produce an emulsion of sample comprisingthe first fluid in the second fluid. Numbered embodiment 130 comprisesthe system of numbered embodiments 127-129 wherein the partitioner isconfigured to produce partitions with an average volume between 0.05 and50 nL. Numbered embodiment 131 comprises the system of numberedembodiments 127-130 wherein the first fluid comprise a dispersed phaseand the second fluid comprises a continuous phase. Numbered embodiment132 comprises a system for conduction of a serial flow emulsionreaction, comprising: a sampling device comprising: a first dispersedphase reservoir; a second dispersed phase reservoir; a third dispersedphase reservoir; optionally, a fourth dispersed phase reservoir; and asampler intake, wherein the sampler intake comprises a first tubeconfigured to puncture a seal of the first dispersed phase reservoir,and a second tube located within the first tube; an injector; a dropletgenerator; a reactor; and a detector, wherein the detector comprises anoptical stage, and wherein the optical stage comprises an opticalrestriction. Numbered embodiment 133 comprises the system of numberedembodiment 132 wherein the first dispersed phase reservoir comprises areaction sample. Numbered embodiment 134 comprises the system ofnumbered embodiments 132-133 wherein the reaction sample comprises anucleic acid molecule, a buffer, a primer, a probe, mastermix, a dNTP,an enzyme, or any combination thereof. Numbered embodiment 135 comprisesthe system of numbered embodiments 132-134 wherein the nucleic acidmolecule comprises RNA or DNA. Numbered embodiment 136 comprises thesystem of numbered embodiments 132-135 wherein the second dispersedphase reservoir comprises a decontamination fluid. Numbered embodiment137 comprises the system of numbered embodiments 132-136 wherein thedecontamination fluid comprises sodium hypochlorite, phosphoric acid,sodium hydroxide, RNAse, or DNAase. Numbered embodiment 138 comprisesthe system of numbered embodiments 132-137 wherein the third dispersedphase reservoir comprises a purge fluid. Numbered embodiment 139comprises the system of numbered embodiments 132-138 wherein the purgefluid comprises water. Numbered embodiment 140 comprises the system ofnumbered embodiments 132-139 wherein the fourth dispersed phasedreservoir comprises a separation fluid. Numbered embodiment 141comprises the system of numbered embodiments 132-140 wherein theseparation fluid comprises an oil. Numbered embodiment 142 comprises thesystem of numbered embodiments 132-141 wherein the sampling devicefurther comprises a staging container. Numbered embodiment 143 comprisesthe system of numbered embodiments 132-142 wherein the staging containercomprises a sample to be analyzed. Numbered embodiment 144 comprises thesystem of numbered embodiments 132-143 wherein the staging container isa microwell plate, a strip of PCR tubes, or a single PCR tube. Numberedembodiment 145 comprises the system of numbered embodiments 132-144wherein the sampling device further comprises a pump. Numberedembodiment 146 comprises the system of numbered embodiments 132-145wherein the pump is a peristaltic pump. Numbered embodiment 147comprises the system of numbered embodiments 132-146 wherein the samplerintake further comprises a double spring configured to position thefirst tube and the second tube. Numbered embodiment 148 comprises thesystem of numbered embodiments 132-147 wherein the first tube of thesampler intake comprises a pointed end. Numbered embodiment 149comprises the system of numbered embodiments 132-148 wherein the firsttube of the sampler intake is a lance, knife, or needle. Numberedembodiment 150 comprises the system of numbered embodiments 132-149wherein the second tube is configured to inject the sample. Numberedembodiment 151 comprises the system of numbered embodiments 132-150wherein the sampler intake is a zero-dead volume injector. Numberedembodiment 152 comprises the system of numbered embodiments 132-151wherein the sampler intake comprises one or more revolvers. Numberedembodiment 153 comprises the system of numbered embodiments 132-152wherein the one or more revolvers inject fluid from the first dispersedphase, the second dispersed phase, the third dispersed phase, the fourthdispersed phase, or combinations thereof. Numbered embodiment 154comprises the system of numbered embodiments 132-153 wherein theinjector is a zero-dead volume injector. Numbered embodiment 155comprises the system of numbered embodiments 132-154 wherein the reactoris a thermal cycler. Numbered embodiment 156 comprises the system ofnumbered embodiments 132-155 wherein the optical restriction is apinhole or slit. Numbered embodiment 157 comprises the system ofnumbered embodiments 132-156 wherein the injector, the dropletgenerator, the reactor, or the detector comprises microfluidic channelsor tubes. Numbered embodiment 158 comprises the system of numberedembodiments 132-157 wherein the microfluidic channels or the tubescomprise one or more connections. Numbered embodiment 159 comprises amethod for conducting a serial flow emulsion reaction, comprising:injecting a first dispersed phase into a flow pathway, wherein the flowpathway is connected to a reactor and one or more detectors; sampling asecond dispersed phase; injecting the second dispersed phase to a wastechannel; injecting a third dispersed phase into the flow pathway;optionally, injecting a fourth dispersed phase into the flow pathway;generating droplets from the first dispersed phase, the third dispersedphase, and optionally the fourth dispersed phase; performing anamplification reaction in the reactor; and detecting a product of theamplification reaction in step (g) by the one or more detectors.Numbered embodiment 160 comprises the method of numbered embodiment 159wherein a first dispersed phase comprises a reaction sample. Numberedembodiment 161 comprises the method of numbered embodiments 159-160wherein the reaction sample comprises a nucleic acid molecule, a buffer,a primer, a probe, mastermix, a dNTP, an enzyme, or any combinationthereof. Numbered embodiment 162 comprises the method of numberedembodiments 159-161 wherein the nucleic acid molecule comprises RNA orDNA. Numbered embodiment 163 comprises the method of numberedembodiments 159-162 wherein the second dispersed phase comprises adecontamination fluid. Numbered embodiment 164 comprises the method ofnumbered embodiments 159-163 wherein the decontamination fluid comprisessodium hypochlorite, phosphoric acid, sodium hydroxide, RNAse, orDNAase. Numbered embodiment 165 comprises the method of numberedembodiments 159-164 wherein the third dispersed phase comprises a purgefluid. Numbered embodiment 166 comprises the method of numberedembodiments 159-165 wherein the purge fluid comprises water. Numberedembodiment 167 comprises the method of numbered embodiments 159-166wherein the fourth dispersed phased comprises a separation fluid.Numbered embodiment 168 comprises the method of numbered embodiments159-167 wherein the separation fluid comprises an oil. Numberedembodiment 169 comprises the method of numbered embodiments 159-168wherein the droplet generator comprises orifices, t-junctions,flow-focusing junctions, or v-junctions. Numbered embodiment 170comprises the method of numbered embodiments 159-169 wherein thedroplets comprise about 0.10 nL to about 1.0 nL. Numbered embodiment 171comprises the method of numbered embodiments 159-170 the dropletscomprise no more than about 0.75 nL. Numbered embodiment 172 comprisesthe method of numbered embodiments 159-171 wherein the reactor is athermal cycler. Numbered embodiment 173 comprise the method of numberedembodiments 159-172 wherein the detector comprises an opticalrestriction. Numbered embodiment 174 comprises the method of numberedembodiments 159-173 wherein the optical restriction is a pinhole orslit. Numbered embodiment 175 comprises the method of numberedembodiments 159-174 wherein the one or more detector comprises one ormore channels. Numbered embodiment 176 comprises the method of numberedembodiments 159-175 wherein the one or more channels are configured todetect one or more wavelengths. Numbered embodiment 177 comprises themethod of numbered embodiments 159-176 wherein the one or more detectorsare arranged spatially. Numbered embodiment 178 comprises the method ofnumbered embodiments 159-177 further comprising introducing a continuousphase following step (g). Numbered embodiment 179 comprises the methodof numbered embodiments 159-178 wherein a channel is narrowed followingintroducing a continuous phase. Numbered embodiment 180 comprises themethod of numbered embodiments 159-179 wherein a frequency ofamplification in the droplets generated not comprising nucleic acids isat most 10%. Numbered embodiment 181 comprises the method of numberedembodiments 159-180 wherein a rate of false amplification is about1:1000 droplets. Numbered embodiment 182 comprises the method ofnumbered embodiments 159-181 wherein a frequency of amplification in thedroplets generated from the third dispersed phase and the fourthdispersed phase is at most 10%. Numbered embodiment 183 comprises anapparatus comprising a sampler intake comprising a first tube configuredto puncture a seal; a second tube located within the first tube; and adouble spring. Numbered embodiment 184 comprises the apparatus ofnumbered embodiment 183 wherein the first tube comprises a pointed end.Numbered embodiment 185 comprises the apparatus of numbered embodiments183-184 wherein the first tube of the sampler intake is a lance, knife,or needle. Numbered embodiment 186 comprises the apparatus of numberedembodiments 183-185 wherein the sampler intake is a zero-dead volumeinjector. Numbered embodiment 187 comprises the apparatus of numberedembodiments 183-186 wherein the double spring comprises a first springand a second spring. Numbered embodiment 188 comprises the apparatus ofnumbered embodiments 183-187 wherein the double spring provides movementin a x, y, or z direction.

1. An apparatus comprising (i) an injector wherein the injectorcomprises an injector conduit; (ii) an intake system wherein the intakesystem comprises a source of a first fluid wherein the source of thefirst fluid can be in fluid communication with the injector conduit viaa first intake conduit when the injector is in a first configuration;(iii) a process system comprising (a) a source of a second fluid,substantially immiscible with the first fluid, wherein the source of thesecond fluid can be in fluid communication with the injector conduit viaa first process conduit and the bulk of the process system via a secondprocess conduit when the injector is in the second configuration, and(b) a partitioner fluidly connected to a third process conduit, whereinthe third process conduit is in fluid communication with the injectorconduit when the injector is in the second configuration, wherein whenthe injector is in the first configuration it can be in fluidcommunication with the first intake conduit but not with the first orsecond process conduits and when the injector is in the secondconfiguration it can be in fluid communication with the first and/orsecond process conduits but not with the first intake conduit.
 2. Theapparatus of claim 1 wherein the partitioner comprises a firstpartitioner inlet fluidly connected to the third process conduit and asecond partitioner inlet fluidly connected to a source of a third fluidsubstantially immiscible with the first fluid.
 3. The apparatus of claim2 wherein the partitioner is configured to produce a mixture comprisinga plurality of partitions of the first fluid in the third fluid, and toflow the mixture out an outlet of the partitioner.
 4. The apparatus ofclaim 1 wherein the partitioner is electrically grounded.
 5. Theapparatus of claim 1 wherein an outlet of the partitioner is fluidlyconnected to (vi) a detector, wherein the detector is configured todetect one or more characteristics of at least one component of at leasta portion of the plurality of partitions created by the partitioner. 6.The apparatus of claim 5 wherein the detector comprises (a) a source ofelectromagnetic energy to provide electromagnetic energy to aninterrogation region of a detector conduit, wherein the interrogationregion is configured for single file movement of partitions through theregion, (b) a detection element to detect electromagnetic energy emittedfrom a partition in the interrogation region; and (c) an opticalrestriction configured and positioned between the interrogation regionand the detection element so that the amount of the electromagneticradiation incident on the detection element is reduced to a fraction ofthe electromagnetic radiation that would be incident on the detectionelement in the absence of the optical restriction.
 7. The apparatus ofclaim 6 wherein the system is configured for lock-in amplification. 8.The apparatus of claim 1 wherein the partitioner is fluidly connected toa reactor, wherein the reactor comprises a source of energy to initiateand/or modulate one or more physical or chemical reactions in one ormore partitions produced by the partitioner.
 9. The apparatus of claim 8wherein the reactor comprises a conduit in fluid communication with thepartitioner, wherein the partitions produced by the partitioner flowthrough the reactor.
 10. The apparatus of claim 9 wherein the reactorcomprises at least two zones, where each zone is maintained at asubstantially uniform temperature, wherein the partitions transit eachof the at least two zones one or more times such that the temperature ofthe partitions transiting each zone is substantially the same as thetemperature of the respective zone being transited.
 11. The apparatus ofclaim 1 wherein the first intake conduit comprises an opening, to beimmersed in the first fluid, wherein a major dimension of the opening isless than a characteristic dimension of any other conduit in theapparatus through which the first fluid will flow.
 12. The apparatus ofclaim 1 wherein at least 95% of inner surfaces of sections of the firstintake conduit, second process conduit, injector conduit, thepartitioner, or any combination thereof, through which the first fluidflows, have a higher affinity for the second fluid than for the firstfluid.
 13. The apparatus of claim 1 wherein the injector conduit has avolume of 2 to 200 μl.
 14. A method comprising (i) providing anapparatus comprising (a) an injector wherein the injector comprises aninjector conduit; (b) an intake system wherein the intake systemcomprises a source of a first fluid wherein the source of the firstfluid can be in fluid communication with the injector conduit via afirst intake conduit when the injector is in a first configuration; (c)a process system comprising (1) a source of a second fluid,substantially immiscible with the first fluid, wherein the source of thesecond fluid can be in fluid communication with the injector conduit viaa first process conduit and the bulk of the process system via a secondprocess conduit when the injector is in the second configuration, and(2) a partitioner fluidly connected to a third process conduit, whereinthe third process conduit is in fluid communication with the injectorconduit when the injector is in the second configuration, wherein whenthe injector is in the first configuration it can be in fluidcommunication with the first intake conduit but not with the first orsecond process conduits and when the injector is in the secondconfiguration it can be in fluid communication with the first and/orsecond process conduits but not with the first intake conduit; (ii)flowing the first fluid from the source of the first fluid through thefirst intake conduit and into the injector conduit when the injector isin the first configuration so that the first fluid occupies at leastpart of the injector conduit; (iii) repositioning the injector to thesecond configuration; (iv) flowing the second fluid from the source ofthe second fluid through the first process conduit into the injectorconduit and through the injector conduit into the process system via thesecond process conduit to displace the first fluid from the firstinjector conduit into the third process conduit; and (v) flowing thefirst fluid through the third process conduit into the partitioner topartition the first fluid into a plurality of partitions.
 15. The methodof 14 wherein the partitioning comprises flowing a third fluid,substantially immiscible with the first fluid from a source of the thirdfluid via a fourth process conduit into the partitioner to partition thefirst fluid into a plurality of partitions of the first fluid in thethird fluid.
 16. The method of claim 14 wherein the partitioner iselectrically grounded.
 17. The method of claim 14 wherein an outlet ofthe partitioner is fluidly connected to (vi) a detector, wherein thedetector is configured to detect one or more characteristics of at leastone component of at least a portion of the plurality of partitionscreated by the partitioner.
 18. The method of claim 14 wherein flowingthe first fluid from the source of the first fluid comprises immersingan opening of the first intake conduit in the first fluid, wherein amajor dimension of the opening is less than a characteristic dimensionof any other conduit in the apparatus through which the first fluid willflow.
 19. The method of claim 14 further comprising flowing thepartitions to a reactor, wherein the reactor comprises a source ofenergy to initiate and/or modulate one or more physical or chemicalreactions in one or more partitions produced by the partitioner.
 20. Anapparatus comprising (i) an injector wherein the injector comprises aninjector conduit wherein the injector is configured to be in fluidcommunication with an first intake conduit for at least part of the timeand allow flow of a first fluid from a source of the first fluid intothe injector conduit via the first intake conduit and to be in fluidcommunication with a first process conduit for at least part of the timeand allow flow of the first fluid from the injection conduit into aprocess system via the first process conduit; (ii) an intake systemcomprising the source of first fluid and a second intake conduitconfigured to be in fluid communication with the first intake conduitfor at least part of the time, wherein the second intake conduitcomprises an opening to be immersed in the first fluid, wherein a majordimension of the opening is less than a characteristic dimension of anyother conduit in the apparatus through which the first fluid will flow;and (iii) the process system comprising a partitioner fluidly connectedto a second process conduit, wherein the second process conduit isconfigured to be in fluid communication with the injector conduit viathe first process conduit for at least part of the time, and wherein thepartitioner is configured to produce a plurality of partitions of thefirst fluid.
 21. The apparatus of claim 20 wherein the source of firstfluid comprises a unit that comprises a plurality of separatecompartments to separate and contain a plurality of samples comprisingthe first fluid.
 22. The apparatus of claim 20 wherein the partitioneris electrically grounded.
 23. The apparatus of claim 20 wherein anoutlet of the partitioner is fluidly connected to a detector, whereinthe detector is configured to detect one or more characteristics of atleast one component of at least a portion of the plurality of partitionscreated by the partitioner
 24. The apparatus of claim 20 furthercomprising a reactor fluidly connected to the partitioner, wherein thereactor comprises a source of energy to initiate and/or modulate one ormore physical or chemical reactions in one or more partitions producedby the partitioner.
 25. The apparatus of claim 24 wherein the reactorcomprises at least two zones, wherein each zone is maintained at asubstantially uniform temperature, and wherein the partitions transiteach of the at least two zones one or more times such that thetemperature of the partitions transiting each zone is substantially thesame as the temperature of the respective zone being transited.
 26. Amethod comprising (i) providing an apparatus comprising (a) an injectorwherein the injector comprises an injector conduit wherein the injectoris configured to be in fluid communication with an first intake conduitfor at least part of the time and allow flow of a first fluid from asource of the first fluid into the injector conduit via the first intakeconduit and to be in fluid communication with a first process conduitfor at least part of the time and allow flow of the first fluid from theinjection conduit into a process system via the first process conduit,(b) an intake system comprising the source of first fluid and a secondintake conduit configured to be in fluid communication with the firstintake conduit for at least part of the time, wherein the second intakeconduit comprises an opening to be immersed in the first fluid, whereina major dimension of the opening is less than a characteristic dimensionof any other conduit in the apparatus through which the first fluid willflow, and (c) the process system comprising a partitioner fluidlyconnected to a second process conduit, wherein the second processconduit is configured to be in fluid communication with the injectorconduit via the first process conduit for at least part of the time, andwherein the partitioner is configured to produce a plurality ofpartitions of the first fluid (ii) flowing the first fluid from thesource of the first fluid through the opening of the second intakeconduit, through the first intake conduit, and into the injectorconduit; (iii) flowing the first fluid from the injector conduit intothe process system via the first process conduit; and (iv) flowing thefirst fluid through the second process conduit into the partitioner. 27.The method of claim 26 wherein the partitioner is electrically groundedfor at least part of the time.
 28. The method of claim 26 furthercomprising flowing the partitions from an outlet of the partitioner to adetector, wherein the detector is configured to detect one or morecharacteristics of at least one component of at least a portion of theplurality of partitions created by the partitioner.
 29. The method ofclaim 26 further flowing the partitions from the partitioner to areactor fluidly connected to the partitioner, wherein the reactorcomprises a source of energy to initiate and/or modulate one or morephysical or chemical reactions in one or more partitions produced by thepartitioner.
 30. The method of claim 26 wherein the reactor comprises atleast two zones, where each zone is maintained at a substantiallyuniform temperature, and transiting the partitions through each of theat least two zones one or more time.